Karpath's note to his son, Peculier in Shadow of Israphel Part 9.
Karpath: Son, if you are reading this, then I am dead. I have walked through the portal to seal it shut. Know that I died to protect this land from a terrible evil. In the Nether is a spirit, who would destroy the world. It must not be allowed into Minecraftia at any costs! If it somehow gets loose once again, you must vanquish it. My armour and blade are here. It falls to you and those that stand with you to save the world. K. Peculier. Fight well, my son. And know... that I love you...*cue a small cutscene of Peculier retrieving his father's blade and putting his armour on.*
Isabel's tale in Part 24. You might find a tear or two in your eyes or on your cheek from how incredibly powerful and moving the tale is. It also makes Isabel and Peculier, who is her brother, both woobies.
Even more so when you consider that Isabel probably thought her brother way plain crazy. All these years and all she saw was him driving himself mad, which was probably at least part of why she ran away in the first place. And then she discovered the truth and probably couldn't find him again to tell him she was sorry. And now she'll never have the chance.
Poor Peculier in Part 34... He through a lot of Break the Cutie in this episode... Finding out that his love, Daisy, is going to be a human sacrifice to the Sand God, believing that he has failed and his father and grandfather are disappointed in him... Yikes... It becomes even more of a Tear Jerker when Simon and Lewis cheer him up... *sniff*
Knight_Peculier: We are too late...it is done. The evil that my father and my grandfather died to seal away is now free again...I have failed. [Knight_Peculier remembers they have three days] Knight_Peculier: Wait...three days? Enough to cross half the world! Enough to seal away an ancient god? With nothing but a sword and this accursed broken map...
Episode 35 has Peculier relaying his past to the heroes. When they first found him he was trying to kill himself, and considered poisoning them with the milk before realizing they were uncorrupted.
Knight Peculier's death in Shadow of Israphel episode 38. Made even worse by the reaction of Simon and Lewis. Yeah, at first they're being their usual selves (you can tell they're trying to keep up their usual irreverent, jocular behaviour and they partly succeed), but they're most likely genuinely shocked.
On that note, Simon and Lewis realizing that, with KP gone, they're completely screwed.
Simon: There's still Daisy to think of, and the world of Minecraftia.
The fact that Lewis immediately tried to backtrack onto the pitch dark, monster filled railway to see if he could see anything, followed by an alarmed (and genuine sounding) 'Oh... shit!' when Simon reminds him of the foreshadowing about their maybe having to leave Knight Peculier behind, does not help.
Not only that but it was so sudden. Not even a proper goodbye or anything. And it wasn't a blaze of glory against a horde of foes, it was a minecart trap and radioactive lava. He didn't even reach Israphel, and what stings even more is that if he had picked a different minecart it wouldn't have happened.
In Episode 41, the duo come across a pool of green lava with a minecart track over it, not unlike where Knight_Peculier died, and are legitimately heartbroken by the sight of it.
Other Minecraft stuff:
Days 3 and 4 of the Advent Calendar have Israphel destroying Simon's Santa grotto and the Christmas tree blowing up.
In the first episode of Rythian's next season, the castle has fallen into disrepair, and poor Rythian clearly sounds depressed.
Also, the silence that falls whenever he looks at something Zoey wrote is heartbreaking. In the fourth episode, he begins talking to himself, expressing his lack of trust in anyone and threatening to kill Duncan.
Season 2, Episode 5. Rythian comes upon the main computer to B.A.R.R.Y. just as Zoey types a message asking if he's there into it. They have a short conversation over Mush Net, and when Zoey says she has to go, Rythian says he has one more thing to tell her. He types 'i miss you', then erases it and replaces it with 'i need you' and erases that and replace it with 'i love you'. As soon as he sends the message, the computer overloads and explodes, without sending the message.
Season 2, Episode 17. It ends with Zoey crying out for Rythian seconds before the nuke goes off, unconsciously repeating the same thing he typed into Mushnet: I miss you/I need you. The bomb goes off before she can say I love you.
Rythian's entire Sanity Slip without Zoey is just heartbreaking.
No matter how she acts Zoeya is clearly traumatised by her experiences. Something made painfully obvious with her unusually nervous reaction to gunfire and a Magical Crafting Table.
Near the end of episode 4, Rythian has to tell Zoey not to get her hopes up about Blackrock, because she's convinced herself that Fishton will be sitting on a chair in their living room, because she thinks that Blackrock is perfectly intact.
Also, there's something sad about Ravs' reaction to seeing Rythian and Zoey again - he's convinced that they "died months ago" (referring to the nuke) and when he first sees Rythian, he asks "Am I hallucinating you again?" Also, when they first spot him, he's playing the lever game by himself. Something about that is saddening. It doesn't last long however.
Look at the placement of the cobwebs. It's in places that he's last seen his friends, and likely where he's been hallucinating them; Teep's chair across from the lever is most noticeably unused.
Zoeya's reaction to the Blackrock crater. She's obviously guilt tripping like crazy about her role in the building's destruction while Rythian tries desperately to simulate a reassuring hug. Minecraft Avatars have never seemed to limiting before.
The Minecraft Christmas song has a moment or two, about a guy who lost his job, isn't able to meet up with his friends and isn't talking to his parents as he thinks they don't like him. This trope then gets Played for Laughs when his Snow Golem melts.
Of course, this all turns into happy tears by the last verse and chorus. His dad texts him to wish him Merry Christmas and proves he does love him after all, and then his friends reveal they've just been waiting for him to come along.
In "Sjin's Farm", Abel losing his parents to Strawfingers.
The ending of Yoglabs Clone Labs, all the way. The episode previous ended on a cliffhanger, so everyone knew something was going down, but nobody expected this. It's shown that Yoglabs controls all the respawning in Minecraft by having one master clone that they just copy from and send out a new clone, but Simon's master clone is killed. In order to keep his ability to respawn, he has to replace it—but in doing so he'll lose his memory. At the end, he and Lewis have a surprisingly heartbreaking farewell before Simon is frozen, and Lewis walks off alone.
The destruction of Duncan's castle at the end of the apprentice and the start of Galacticraft is downplayed, but still hits quite hard for those who've seen Duncan's tekkit series. The castle was second only to Sjin's farm in scale, and had even more valuable resources and advanced machines inside it, after all the work Duncan put in, and then it is completely ruined in an instant
The aftermath of the accidental destruction of the Jaffa Factory in Jaffaquest 106. It hits even harder because the ending of the previous episode was hilarious. It's very sobering to tune in and see the amount of devastation that occurred.
Simon's Suddenly Shouting and ordering Lewis and Duncan to pack up everything only highlights just how hard he'd been hit by the mistake.
In episode 17 of Flux Buddies Kim and Duncan continue their raid on the Dwarven base, which they deemed evil due to all the stereotypical tells (rooms made of Netherack, lava, darkness, etc). Except when they open the chests, expecting to find great loot just like in the previous dungeon... all they find are food supplies. Meanwhile the dwarves are chatting and hanging around and only attack when provoked. The two of them spend the rest of the raid guilt tripping.
In Episode 25 of Flux Buddies, Duncan and Kim are trying to move their cat, Tiddles II, into a new home, and they do so by using Nether Portals. They land in a house, and Duncan can only watch as Tiddles II walks off the platform and falls into the lava, dying instantly. The fact that Kim chews Duncan out for it only makes it worse.