The "Kenny Dies" episode, where he is Killed Off for Real... not in the usual over-the-top fashion, but slowly, due to a debilitating illness, with the episode's main plot revolving around his friends trying to get embryonic stem cell research legalized in the hope that a treatment can be developed before Kenny dies. The writers eventually brought him back anyway, after which he started dying very rarely. Many of the scenes in the episode are genuinely heartfelt and Played for Drama.
It's played absurdly straight in the "Coon and Friends" saga. Kenny, who is revealed to be the real Mysterion, has stated that his superpower is that he cannot die, and that even when he does die, no-one ever remembers it happening to him (itself a bit of a Retcon, since they sometimes did in the early seasons). He hates it so much that his near-Heroic Sacrifice at the end comes off like a desperate attempt to die for real. One might even see it as a Deconstruction of Negative Continuity. Also, this ability apparently comes from his parents having been involved with the Cult of Cthulhu.
In the episode "City Sushi", it's revealed Mr. Kim is an insane Caucasian psychiatrist with multiple personalities. By the end of the episode, everyone allows the Mr. Kim personality to take over completely, since curing him would cost the town its only Asian restaurant.
The two-part Wham Episode "You're Getting Old"/"Assburgers" reveals that all of Randy's wacky hijinks come from the fact that he's unhappy with his life and trying to find something that will fix the problem.
It might have always been there but "Cash For Gold" revealed that Stan's grandpa has Alzheimer's disease, making him calling Stan "Billy" all those times a little less funny.
In South Park, there is a show known as Terrence and Phillip. The show is rather goofy in nature, but for some strange reason Ike just so happened to resemble the characters on that show instead of looking more like Kyle and his parents. However, the show initially took little notice of this, instead using Kyle's football-shaped head as a joke by having Ike kick his baby brother despite Ike telling him not to do so. "Don't kick the baby!" However, the authors themselves eventually noticed this, and they revealed that Ike was adopted and was not in fact biologically related to Kyle as previously believed, and that he was Canadian just like Terrence and Phillip.
In The Venture Bros., how Master Billy Quizboy got his mechanical hand is told through The Rashomon, and the end of the episode has him saying he doesn't really remember. A later episode shows this was because he had his mind wiped. His arm was bitten off by a pit-bull, and was given the replacement which had a monitoring device so he could act as The Mole.
Helga's family life and her bullying ways in Hey Arnold! was originally played for laughs (her dad is a pompous jerk who thinks everyone should bow down to him, her mom is a scatter-brained smoothie drinker, and her sister is a Mary Sue who doesn't understand how cruel life can be, and Helga acts like a bully because she's hiding her true feelings for Arnold) — until the "Helga on the Couch" episode reveals that she's been neglected by her parents for years and Arnold actually was nice to her on her first day of pre-school. To top it off, Craig Bartlett (the show creator) has stated in many interviews that Helga's mom is an alcoholic, but, because Nickelodeon censors didn't want alcoholism to be mentioned on the show, the "smoothies" were created as a means to let her alcoholism slip past the censors. There are also the other references to Miriam being a drunk: she once did court-ordered community service for a crime she doesn't remember doing, she falls asleep behind the couch, she had her driver's license revoked, and she tries to sober up by drinking coffee (which is actually a myth).
In the Family Guy episode "Jerome Is the New Black", Quagmire tells Brian that Cheryl Tiegs was the love of his life who left him and the break-up is the reason why Quagmire is a sex addict. Three past episodes had clues that make the revelation logical (and not just something the writers pulled out of their butts): in "Emission Impossible," Quagmire has a poster of Cheryl Tiegs on his refrigerator (during the scene where Chris shows Quagmire the objects he found on the scavenger hunt), in "The Perfect Castaway," Quagmire's reason for wanting to be blind (in a game the guys are playing) is because every woman he has sex with will, in his mind, be like Cheryl Tiegs, and in "Barely Legal" (the one where Meg becomes obsessed with Brian), Quagmire gives Meg the Shel Silverstein book The Missing Piece and tells her that he reads it whenever he feels that he needs to find the one thing in his life that's missing.
"Jerome is the New Black" also had a gag involving Quagmire's sister and her abusive boyfriend, which was played for tragic laughs (as Brian thought Quagmire's battered sister was just some one-night stand Quagmire had rough sex with). Two seasons later in "Screams of Silence: The Story of Brenda Q.", Quagmire's sister and her abusive boyfriend come back, and the abuse is Played for Drama.
In the episode "Yug Ylimaf" (the 200th episode where Stewie and Brian accidentally cause time to start going backwards), we see why the Greased-up Deaf Guy (a gag character who first appeared on the episode "The Thin White Line") is the way he is because he was walking beside a grease truck which suddenly exploded, burning away his clothes and leaving him soaked in grease and deafened by the explosion.
Parodied in a Cutaway Gag from "Meg Stinks!" which shows how Peter funds all his wacky schemes: armed robbery!
"Where The Buggalo Roam" features a gag where Kif believes that kissing Amy is the same as making love to her. A later episode reveals that Kif's Bizarre Alien Biology causes his skin to become receptive to genetic material whenever he feels a great sense of love for someone else. In other words, kissing Amy really is making love to her.
In a season one episode, for a bit gag, Amy's parents set her up with Kif. In the season three premiere, we learn that Kif has been hopelessly pining for Amy since then but has been too scared to ask her out again.
The first episode makes Fry's life in the year 2000 seem utterly miserable, so that it's understandable how he celebrates after being unfrozen a millennium later. Since then episodes have gone back and explored his previous life more closely, creating drama as Fry remembers his family, his girlfriend, and, most famously, his beloved dog.
Mutants were shown to live in the sewers in one episode, and a Running Gag developed where they would stick their heads out of the ground to yell at people. The mutants' situation is Played for Drama later, when it's revealed that Leela is a mutant, whose parents gave her up so she could pass as an alien and live on the surface.
In "Teenage Mutant Leela's Hurdles", when the cast is de-aged to teenagers, teenage Amy is shown as fat and her father makes fun of her weight. In "The Prisoner of Benda," it's revealed that Amy was a compulsive over-eater, which is why she was fat as a kid.
Parodied in "Behind the Laughter", the outside-of-canon Animated Actors episode in which we're told that Homer became addicted to painkillers after falling down Springfield Gorge (in a well-known early episode), and that that enabled him to do "the bone-cracking physical comedy that made him a star." There is also, in the same episode, Homer feeding Lisa and Bart growth stunters via executive order in order to keep them looking the same age for the show to explain them never aging.
In the episode "Eternal Moonshine of the Simpson Mind", Homer has his life flash before his eyes. The dysfunctional character that we have come to know and love is shown to have a pretty poor time growing up, which makes the dark humor in the show harder to swallow.
It may have been implied in the past; but the Season 27 episode "Puffless" reveals that Patty and Selma's father died of lung cancer when they were young. This makes his appearance in the Season 2 episode "The Way We Was" (where he is shown smoking a cigar and declaring that Marge meeting Homer "took years off his life") and subsequent mentions after that far less amusing.
Eleanor Abernathy aka "the Crazy Cat Lady" was first introduced in Season 9's "Girly Edition", as Lisa was trying to compete with Bart's sappy human interest stories. In Season 18's "Springfield Up", we see her early life. At age 8, she was a bright student who was already aspiring to be both a doctor and a lawyer, and by age 24, got her degrees for both fields. But by age 32, she was already beginning to feel burned out, and began drinking and acquired her first cat, giggling a little maniacally at that point. Skipping ahead to age 40, we see that she's become the broken/disheveled cat lady shrieking and wailing incomprehensibly.
The Season 22 episode "Moms I'd Like to Forget" has a flashback scene where Clancy Wiggum is holding baby Ralph in his arms, then accidentally drops him on his head. Clancy quickly picks the baby up, but notices that Ralph's head has been slightly deformed, and his mannerisms have changed. So it is strongly implied here that Chief Wiggum's clumsiness is the reason for his son's quirks and mental deficiencies.
Most of the third season of Moral Orel does this to the first season. The first season is a comedy and Orel's life is portrayed as a Hilariously Abusive Childhood. Despite Orel's parents being pretty bad people, Orel is quite cheerful for the first two seasons, which might have made the implications of this less noticeable. It's towards the end of the second season where Orel realizes his father isn't the awesome guy he thought he was. It might be a Cerebus Retcon for Orel himself as well.
The "Lost Commandments" in particular are given a specific explanation. At first just seeming like a general jab at Christianity, it turns out that the entire thing is a Puppington family tradition, going at least as far back as Clay's mother.
Early episodes have the Ice King portrayed as a comedic Psychopathic Man Child with a lust for women (particularly princesses) much younger than him that was often Played for Laughs. Come the episode "Holly Jolly Secrets": In a pre-apocalypse Earth, the Ice King was an intelligent, mild mannered antique dealer named Simon Petrikov who bought an ancient magic Scandinavian crown. After putting it on as a joke in front of his fiancée (whom he affectionately referred to as "My Princess"), he did something he couldn't remember that drove her away forever. The Crown soon seized his mind and began altering his body, giving him power over ice and snow. In the process, he heard voices and had constant visions that drove him utterly and completely mad until he became the Ice King we know today. The Ice King also no longer seems to remember or understand what's happened to him. Made lighter by "Betty" where the reason his fiancee left him is revealed to be him, or more specifically, himself from 1000 or so years in the future after the Ice Crown has its powers and effects negated when an anti-magic being is summoned.
In "Evicted" Marceline suddenly reveals herself as the past owner of Finn's and Jake's home and kicks them out, which is used as the starting point for a comic adventure. In "I Remember You", it's revealed that the reason why she has lived in so many different places is because Simon/Ice King as above was her foster father at the time that he was declining into madness, and that she keeps moving because he keeps instinctively looking for her without remembering their relationship, and acts either pathetic or dangerous in manners that she couldn't bear.
In "Ocarina", it's revealed that the rare occasions that Jake's children have appeared over the last season or so are in fact the only times he's ever spoken to them, because he's embarrassed that due to their Rapid Aging they're more emotionally mature than he is, and that at least one of them absolutely hates him as a result.
A minor one: In "Everything Stays", it is revealed that Finn, Susan Strong, and the various mutated humans living underground wear animal hats because in the years following the Mushroom War, wearing them was a method of camouflage and protection against vampires. Even after Marceline managed to kill all of them, the notion stuck for generations afterwards.
In the same show, Fred's status as a "Well Done, Son!" Guy gets this treatment. In Season 1, his relationship with his emotionally distant father was Played for Laughs, through the obvious contrast between the relentlessly optimistic teenage nerd and his uptight politician father. But it gets a lot less funny after the first season's finale reveals that Fred Jones Sr. actually kidnapped an infant Fred Jr. from his real parents, and he has essentially been keeping him as a hostage for his entire life. Becomes more complicated when it's discovered that his birth parents, Brad and Judy were antagonistic, having become influenced by the Disk Pieces they were after. However, it is revealed that many of their positive qualities were repressed by the Greater-Scope Villain and it's revealed that the mayor really did care for Fred and he was, in a way, a better parent than his birth-parents (least before the World-Healing Wave.)
In general, the classic Scooby-Doo formula that we all know and love—criminals pose as monsters to pull off overly complex schemes before being unmasked by the kids—has never really been questioned, and it's just an accepted convention that the Scooby-Doo universe is filled with crooks who all decide to pose as monsters for their own unrelated reasons. Mystery Incorporated's complex Myth Arc finally explores the phenomenon, revealing that it's not such a coincidence after all: all of the Gang's past adversaries have been the Unwitting Pawns of a malevolent Eldritch Abomination that compelled them to embrace their darker sides by taking the forms of monsters. On top of that, we learn that at least some of them (notably Fred Jones, Sr.) were inspired to adopt their monstrous alter-egos by dreams of real monsters from an alternate dimension.
Spike was often the Butt-Monkey of earlier seasons, with season 3 having several entire episodes revolving around him hilariously failing at things. Starting with season 4, Spike has a character arc revolving around his self-esteem issues and generally feeling useless.
Early in the very first episode, Twilight rudely declines an invitation to an unseen friend's party to focus on her studies. It's treated as a quick Establishing Character Moment for her anti-social attitude, and is all but forgotten when Twilight moves away and makes new friends. Five seasons later in "Amending Fences", Twilight finds out that her friend—who already suffered from social anxiety and low self-esteem—degenerated to become a bitter and lonely shut-in, all because one of her so-called friends didn't turn up and eventually forgot all about her. It also serves as a reflection of the kind of pony Twilight could have become had she never made friends.
"Brotherhooves Social" provides an example of what kind of effect the Mane Six's absurd amount of series-wide accomplishments (which include saving Equestria on a semi-regular basis) would have on others. Here we see that Applejack is considered the family hero, but her older brother Big Macintosh feels somewhat resentful for getting ignored by their little sister Apple Bloom, who used to idolize him before Applejack started stealing his thunder. The fact that he rarely opens up to speak his mind suggests that he's been stewing over this longer than he'd care to admit.
Fan favorite Derpy Hooves, of all ponies, ended up being on the receiving end of this. She initially became popular due to her Fish Eyes making her visually distinct from every other pony in the show. Her eyes and klutziness were used mainly for humor and cuteness... until a Meaningful Background Event in "Parental Glideance". In that episode, a flashback showing Rainbow Dash placing higher and higher in flying competitions also shows Derpy, who starts out at the top of the podium with normal eyes and performs progressively worse and worse as her eyes become more misaligned, strongly implying that she could've rivaled Rainbow Dash in flying ability if not for her eye problems. Adding to this is another flashback in "Where the Apple Lies" showing a slightly older Derpy in the hospital with with bandages on her eyes, implying a failed attempt at fixing them.
Throughout the course of Ed, Edd n Eddy, Eddy was portrayed as a Small Name, Big Egoloud mouthscam artist who looks up to his brother and whose schemes usually go hilariously wrong and generally got his comeuppance at the end of the episode, often by being beaten up by one or several of the Cul de Sac kids. It's all Played for Laughs... and then the movie comes along, where it's revealed that Eddy's brother was a Big Brother Bully who regularly beat up Eddy, and his Small Name, Big Ego traits were the result of an Inferiority Superiority Complex he developed from this. His whole scamming and loud mouth nature were the result of trying to imitate his brother, whom he thought the other Cul de Sac kids respected. (When in reality, they were all terrified of him.) Knowing this, it makes a lot of the earlier episodes, especially the ones where Eddy suffers Disproportionate Retribution, very hard to watch.
In the Earthworm Jim episode "Queen What's-Her-Name" we learn that Princess What's-Her-Name is called so because she was such The Unfavorite that no one bothered giving her a proper name at all.
Archer's fear of alligators. It was first introduced and Played for Laughs in "Pipeline Fever"; understandably, the other characters found it amusing that a reckless adrenaline junkie could be reduced to quivering fits by the mere mention of an animal. Two years later, "Once Bitten" revealed the origin of his phobia: he actually loved alligators as a child, but one of his last (and only) memories of his biological father was of him bringing him a stuffed toy alligator as a present. Archer apparently finds that memory so troubling that it takes a near-death flashback to bring it back.
In the episode "Steven's Lion", when discussing whether Steven should be allowed to keep Lion, Garnet remarks that they "kept Amethyst", which is treated as a joke by Pearl. To the audience, it seems to be an innocent joke referencing Amethyst's rambunctious nature; however, in the later episode "On the Run", it is revealed that Amethyst was created in a Gem-making facility known as a "Kindergarten", that they actually did keep her, and that learning about her origins gave her a deep sense of self-loathing. Learning that she's also a defective Gem, having spent too long in incubation, in "Too Far" doesn't help.
In "Marble Madness", when Peridot meets Steven and wonders if "Stevens" have supplanted humans as the native species on Earth, Steven starts listing some of the other humans he knows. In the season 4 finale "I Am My Mom", it turns out Homeworld interpreted this as a list of different types of human, and sent Aquamarine and Topaz to kidnap Steven's friends and neighbors to take to the Diamonds' People Zoo.
Beast Wars ends with the heroes victorious and returning home with Megatron strapped to the outside of their space ship in a humiliating fashion. And then comes Darker and EdgierBeast Machines where it's revealed that Megatron was able to escape mid-flight (which of course is made extremely easy when you're strapped to the outside of the ship and thus nobody inside is able to stop you) allowing him to conquer Cybertron before our heroes even arrived.
Throughout the first season, we've seen Star battle monsters who have tried to steal her powerful wand, apparently for some evil goal. Come "Mewnipendence Day", we learn that Mewni's original settlers massacred the monsters and stole their land, while proclaiming themselves to be righteous. While Ludo himself is still treated as a joke, the desire of him (and later, the more threatening Toffee) is now re-framed from being Cartoonish Supervillainy to retribution.
There's also the Running Gag of St. Olga's School for Reformed Princesses, with Star constantly being threatened to be sent there because of her hyperactive nature. When the school is actually revealed to be an Assimilation Academy, the jokes come off as less funny and more genuinely frightening.
Queen Moon's overprotective nature is revealed to be from the fact that she became queen after her mother was killed and wasn't fully prepared to take up her mantle, so she wishes for her daughter to be prepared for such an unfortunate occurrence.
The depiction of Mina Loveberry as a Cloudcuckoolander is later revealed to come from a mix of Super Soldier serum giving her an unnaturally long lifespan and being the only remaining solider from those earliest wars against monsters giving her PTSD.
In the Wander over Yonder episode "The Battle Royale", THE BLACK CUBE OF DARKNESS manages to get to the Ring of Power, but is unable to pick it up because of its lack of hands, and it leaves while a melodramatic sad tune plays. This is played purely for laughs, but a later episode, "The Black Cube" showed that this event caused THE BLACK CUBE to lose all respect as a villain and end up living in a seedy apartment, working a dead-end job as a fast food cashier, and being alternately mocked and feared by his neighbors.
The ending of "The Wanders" does this for Wander's entire philosophy. He doesn't just help because Good Feels Good, he helps because he knows what its like to be helpless. It also implies that he hates that part about himself, since he is very reluctant to merge with his helpless persona.
Rick and Morty: At the end of the episode introducing the Citadel, Morty asks what will happen to all the Mortys who lost their Ricks. He's told that the Rickless Mortys will return home and lead ordinary lives. Instead, "Tales from the Citadel" reveals that Rickless Mortys are kept away from their families and sent to a school where they are groomed to serve as docile replacements for other Ricks, with many shuffling through many Ricks. Mortys who fail to graduate are dumped in "Mortytown," a burnt out, crime infested section of the citadel where they victimize each other.
Iron Man: The first season had an episode where Tony pretended he was going to marry Julia Carpenter as part of a plot to fool the Mandarin. At the time, Julia was clearly in on the ruse, and seemed to have no problem with it. In the second season, Julia is instead shown to be quite bitter about having been left at the altar, and her lingering resentment causes tension with Tony in several episodes.
Avatar: The Last Airbender: All throughout Season 1 and the beginning of Season 2, humorous things happen to Zuko that foil his attempts at catching Aang. At the end of "Bitter Work" (after being unable to create lightning), he's screaming into a storm about how cruel the universe has always been to him and finally breaks down. Suddenly, all those silly things go from the universe poking fun at an Ineffectual Sympathetic Villain to the turmoils of a young man desperate to go home and be loved by his father.
Arguably, the episode "The Beach" can be this for his sister too. Azula fails in spectacular ways to socialize normally like other kids. However, the defection of Zuko and the betrayal of Mai and Ty Lee is a massive blow to her beliefs and philosophies she was trained into believing in despite the normal ways she "converses" through fear and manipulation which begins her Sanity Slippage into a Villainous Breakdown. Suddenly, you look back at this episode and realize that her inability to socialize messed her up far heavier than implied.
G.I. Joe: Resolute: In the original Sunbow cartoon, Cobra Commander was hammy, bumbling, and generally not meant to be taken seriously. Come Resolute he reveals this was all an act to encourage his underlings to act independently. He then takes charge and begins obliterating cities and executing his minions.
Over the course of Transformers: Robots in Disguise the Autobot Council is slow to react to threats facing Earth and respond the heroes requests for assistance. This is initially dismissed as bureaucratic sloth and the heroes manage to prevail on their own. Then Ratchet arrives from Cybertron and reveals that the Council is running a political smear campaign against Optimus Prime and those loyal to him. The series finale reveals that they were actually Decepticons in disguise and their inaction was a ploy to strengthen their grip on the populace before starting a campaign of galactic conquest, starting with Earth.