All of the Seven Deadly Sins mini-bosses have an unique sprite not seen in any other enemy in the game... All of them, except Sloth, which is just a lazypalette swap of the Globins! That's a subtle, yet incredibly clever and ingenious joke.
Also, the worms he spawns that are usually pretty fast when charging are slower than the rest. They're lazy.
This has become even more plausible with the upgraded Sins from Wrath of the Lamb. Each has a disturbing new design... except Super Sloth, who's just a reskin of the Glazing Globins, as regular Sloth is to regular Globins.
The familiar you get from obtaining the Cube of Meat can push your bombs around, which can often spell disaster for you. If you get three Cubes of Meat, he will get his own body and follow you around. However, he'll still push your bombs around. Getting the Fetus in a Jar item makes it so your only attack is bombs. It also gives you a top hat and a monocle, reminiscent of Dr. Fetus. Combining the resulting bomb-spam and Meat Boy will probably kill you, because Meat Boy will often push your own bombs back at you. Could this be Meat Boy trying to get revenge on Dr. Fetus?
Whore of Babylon transforms the character into a demon with high attack power, but only when you have only one heart - AKA when you are near death. ??? does not have regular hearts, ever, therefore maintaining WoB always active. Makes sense, since he is a Dead Blue Baby, and therefore as close to death as possible.
Look at the names of the playable characters: Isaac (a sacrifice to god), Judas (a traitor), Cain (a murderer), Magdelene (a whore), and Eve (the first sinner). Says a lot of what Mom thinks of her kids, doesn't it?
That says more about what Isaac thinks of himself, given that the other characters are Isaac in various costumes...
Another Fridge Brilliance moment comes in the addition of Samson, a man who was given incredible strength by God, and used it to great effect. It seems he's finally snapped out of his depression. The Wrath of the Lamb update in general showed that God has finally stepped into the ring to try and help Isaac escape.
Judas starts with 3 pennies, which seems odd — it'd seem more appropriate for Cain to have them, right? Except the original Judas sold out Jesus for thirty (3*10) coins.
It's more interesting than just "cancer makes you sad". The Cancer trinket has a unique effect in that it increases your rate of fire, without counting as a rate of fire upgrade. This means you can have upgraded your rate of fire to the cap, and Cancer will still increase your rate of fire, implying that no matter how sad you are, cancer can always make your situation worse.
Dad's Key allows you to exit any room, including challenge rooms. Now we know why Isaac only has a mom.
May fit under Fridge Horror, but ??? starts off with "the Poop." At first, I assumed this was purely because ??? is the Lethal Joke Character, so he starts out with the joke item. However, when someone dies, the muscles within their body relax, resulting in a post-mortem bowel evacuation. ??? starts out with Poop because that's actually all he had!
The Whore of Babylon is a curse effect when you drop to half a heart. When Eve ate the fruit and became the first sinner, she was cursed by God and cast out of paradise; this why Eve in game has to carry this curse no matter what.
Envy has a distinctly trollface-like smile, and he also often drops troll bombs on death. Perhaps trolls are envious of what other people have?
All the trinkets you get from Beating Blue Baby with a certain character is something they've lost! Isaac has metaphorically lost his head, Cain is missing an eye, Judas, lost his tongue after his betrayal of Jesus Christ, Maggy has lost her faith, Eve's bird has lost its foot, Blue Baby has lost his soul, and Samson, in the Bible, had a lock of his hair cut off, which made him lose his strength!
Also, when you think about it, Isaac being a sacrifice for his mother means he's most likely thinking about being killed by mother, and thus decapitation. Also the head joins you like other, dead or imaginary, familiars.
Looking closely at Larry Jr. reveals that it's constantly crying. And why not? Its parents were killed back in Super Meat Boy.
Why does having flight allow you into a Curse Room without taking damage, but still hurts you on the way out? It's because the spikes in facing into the room, meaning that Isaac must prick himself trying to squeeze his way back out!
Mulligans will occasionally spawn in a room with fires in it, and almost inevitably they'll end up killing themselves by walking into it. They're too stupid to notice it's there, right? Then you remember that they're people who are being horrifically mutated into hives for insects, and it's clear from their sobs that they don't like it, and you realize that they're probably trying to commit suicide.
Same happens when they're left alone for a while, they'll just decide to blow themselves up.
Which begs the question of why their bomb-carrying variants want to blow you up too. Unless they're so depressed for their existence that they're taking you with them.
The corpse that's in both the shop and hidden rooms has a uncanny resemblance to Greed. At first, seemingly a coincidence. Then the fact that Greed only attacks in these rooms implies that for whatever reason, Greed is playing dead on the occasions he doesn't attack...
Also Fridge Brilliance because his name is Greed, and when he sells you things, he...you know...takes your money.
Which also explains why he has a higher chance of spawning when you've visited a shop before or have a large amount of money, he's trying to take more money from you than you could pay him by buying out his wares.
When Isaac picks up the Steam Sale item, it shows Isaac with a sale tag on his ear. Has Mom been selling Isaac's body to pedophiles as a means of making money?
I doubt it; she's a religious fanatic, and that doesn't fit with that.
Or perhaps it could be a reference to Isaac selling his soul.
There is a between-floor nightmare where Isaac is suffocating in a chest. Beating It Lives for the first time gives you an ending where you find ??? in the chest. ??? is dead. There are a few endings that imply that Isaac went into the chest. Do the math.
The Ankh item resurrects Issac as ??? if he dies. One ending has him dragged into the chest by Mom. You work it out.
To anyone who gives Issac cancer (a trinket, boosts tear rate) and decides not to swap it out when another trinket spawns: You gave a child cancer and neglected to cure it. You monster! This is all the more tempting, given how useful a more tearful Issac is.
Similarly The Curved Horn is a representation of Isaac being demonic or demon possessed. Keeping it over other trinkets means you want a demonic (possessed) child! You monster! Again, a very good item as it increases your damage output considerably.
The SMB Super Fan item basically turns you into Meat Boy. Meat Boy has no skin. The aforementioned item's icon is a circular saw blade. Thinkabout it.
In the final level, the Chest, you can't bomb your way out of rooms. Isaac couldn't escape the chest, no matter how hard he tried.
Items and their effects tend to get this way if you think about them. For instance, the "Wooden Spoon" or "Belt" items makes Isaac faster... because he's running from being spanked! "Squeezy" may have been one of Isaac's favorite toys, so it makes him cry more as he holds it and remembers the good times in life (which is why he also gets two soul hearts). This also means when you get a pill that decreases tear damage/speed/range, you've actually made Isaac take something that makes him feel better, so he's crying less... even though this is portrayed as a negative thing
Think about it. A Tears Down pill is actually an antidepressant, so Isaac cries less; conversely, Tears Up is a depressant! What the Hell, Player??
Then again. Making Isaac cry more means that he's more likely to survive. It's sort of like making someone aware of how much shit he's going through would make them more able to deal with it in the long turn.
On a similar note, Bad Trip is normally just some drug that hurts Isaac. But when he's at 1/2 heart, it becomes Full Health. Isaac's so hurt that giving him the Bad Trip drug makes him hallucinate and more pain resistant, allowing him to keep limping along.
There is a "Max's Head" item that makes Isaac's tears bigger and more powerful... There's also a "Lunch" item that is a dog bowl with the name "Max" on it... no wonder Isaac's tears get stronger because of "Max's Head"... it was his pet dog!
Ipecacis a serum that makes you vomit. It was used to expel poison from the body; however, ipecac itself is poisonous and has many of the same side effects. Moreover, it has been out of use for nearly a century and is in fact recommended to throw it away. Why would Isaac's mom still have this?
The familiars have names like Sister Maggie, Brother Bobby, and the like. Either Mom had multiple miscarriages/abortions, or this isn't the first time "God" demanded a sacrifice...
The Disc One final floor is the Womb, which is basically Isaac's mom's womb. It's not as bad as it sounds, until you realize what sorts of things Isaac's mother actually has in her womb, possibly including a tapeworm, a fallen angel, or the physical representation of death, depending on the RNG's benevolence.
Not only that, but her womb is actually divided into rooms.
One of these rooms can be a library. Isaac's mom literally has a library inside her vagina.
There's also a boss there called Teratoma. Teratomas are tumors with human body structures such as eyes, arms, legs, and teeth, and are generally present from birth and can be passed on from mother to child. Let us remind you that you fight the Teratoma in The Womb.
Take a look at the remains from a destroyed pile of poop. It looks remarkably like a smiley face. Is Isaac playing with the poop and making faces?
There's numerous flies and poop piles in Isaac's basement, the flies are enemies, and the poop is rewardable vandalism material. Wearing an item called skatole, which is poop, deters flies. Yet you can't just grab a poop pile and smear it over yourself.
Maybe the Skatole is a notably solid poop that Isaac can carry around.
What kind of shopkeeper locks the store so that customers can't come in unless they have a key? Talk about being bad for business...
Greed doesn't want ordinary customers. He wants customers so desperate that they will hunt down keys to get to him. You might as well ask why Greed sometimes tries to kill them.
Fridge Brilliance: if you expended a key to enter the shop, you would be more likely to buy something after entering, even if this thing is another key to replace the one you used. You wouldn't want the key to have gone to waste, after all.