Matt's fate in the alternate reality where Edd was never born in "Christmas Special 2005". Whereas Tom ends up as a wealthy director shooting adult films with Tord, who is now a communist dictator, Matt ends up homeless and starving on the streets of London because Edd wasn't there to introduce him to Flash. The sight of him standing there sadly with Puppy-Dog Eyes and a sign begging for handouts is heartbreaking.
Though it can be laughed at, when Tom's friends kill him, throw him in the trash can and replace him with a Matt clone in "Spares" is pretty sad, as it implies that his friends don't care about him at all.
Also doubling as They Changed It, Now It Sucks! for some people, Tord driving off in the beginning of "25ft Under the Seat" and being Put on a Bus for the rest of the series. Even Zanta Claws isn't happy about that, stating that he was his favorite character.
Laurel's death in "MovieMakers", while Played for Laughs, can come off as this. She was a genuine Nice Girl who was actually getting along with the boys and having fun making movies with them. Then she was killed by Shoe. To make it worse, Word of God has confirmed that she was Killed Off for Real and won't be returning.
The "desire room" bit in Space Face is quite lighthearted until the very end of the scene, when Commander Bai stands there, looking sadly at the image of a small planet.
Matt: What's that? Bai: Home. Let's move on.
This was the last line Edd animated. Just after, the boys enter the helm with three lights shining over them...and the light over Edd’s goes out.
Tom spends most of "Space Face: Part 2", the first episode since the real Edd Gould's death, looking sullen with few speaking lines and a drinking flask in hand. This dialogue from the end of the episode has some uncomfortable Reality Subtext considering Tom Ridgewell suffered from severe depression and a drinking problem around this time:
Edd: You know, you've been pretty quiet today, Tom. Tom: I am very drunk.
Similarly, Edd's saddened reaction to the voice changer gun breaking, making it impossible for Matt to give him his old voice back.
A brief one occurs in Tom's Tales of Crazy. In the beginning Tom narrates how the 'Tom's Tales of...' started with the first two installments with two drawings of his and Edd's heads, before jumping on quickly describing asdfmovie and then stating 'stuff got kinda wierd', with Edd's head disappearing and for a small moment, Tom looks saddened.
The End: Part 2 is a huge one:
Watching Tord punch Matt in the face can be a bit hard if you're a Matt fan.
Tom's (failed) search for a new home leaves him disheveled and living in a cardboard box in an alleyway. He tries to reassure himself that everything will be just fine...before bursting into tears. Unlike other times we've seen the boys cry in Eddsworld, this is most definitely not played for laughs.
Jon's death at Tord's hands.. It's not played for laughs, and Eduardo is at the verge of tears as his friend/roommate literally dies in his arms.
Eduardo: Jon? Jon?! Speak to me, buddy! Say something! Say something, you idiot! Jon: Something. [laughs weakly, dies]
In Part 1, Eduardo reacts to Jon annoying him by growling "I wish you were dead!"Be Careful What You Wish For...
Hell, Tord's betrayal of the gang in general is a major downer. Edd was so overjoyed about his old friend coming home, only to realise it was for the wrong reasons...
Even Tord's expression after he's defeated could qualify. He's angry, but as he looks out at Edd, Tom and Matt leaving, there's a hint of melancholy on his face, as if he's realizing that whatever friendship he had with the others is officially gone now, and after what he's done, there's no way for him to get it back. Not helped by the crew revealing at ComiCon London that Tord is very unlikely to come back after the show's revival and that even if he does, it will only be for one episode to give him one last goodbye.
The ending of the episode with while comedic, focuses on Edd "leaving", with only Tom giving a small smile and saying "See ya, Edd", arguably eluding to the real life Tom saying goodbye to Edd himself, who had long passed in 2012.
Adding greater impact to the ending credits where Tom himself confirms the end of the Eddsworld: Legacy campaign and not only passing over the show once more to Edd's family, but his own departure from the show as well and bidding the show farewell. This, along with the departure of Edd's voice actor at the moment, Tim Hautekiet, from the show. And it doesn't help that Edd's lines seem to resemble how while Tim wasn't always there, he had fun working for Eddsworld, but it was time for him to move on.
"The Beaster Bunny" reveals that Eduardo's still depressed about Jon's death, to the point where he’s apparently become somewhat a shut-in. The cartoon is also the first one since the very early days to not have Tom voice Tom, with a new VA taking the role. Matt is the only one still voiced by the original crew.
On a similar note, WTFuture was one of the very last episodes where the entire original voice cast of the main four was all together (Tord Larsson made a cameo re-recording an old line from a previous episode).
. The comic shows that he is not happy about her death.