Claudus' death. It was right after a moment of triumph and a Moment Of Awesome. It was rather unexpected, especially because his voice actor voiced Lion-O in the original series and had been making his rounds on the internet, Comic-con, and Cartoon Network to promote the new series.
Made worse by the fact that he died believing that both his best friends- Grune and Panthro- had betrayed him, not realizing that Panthro was being impersonated by Mumm-Ra.
Also, The Cleric Jaga's apparent death. The pure awesomeness and wisdom the character showed in only one episode is amazing. At least he died fighting. Those familiar with the character, however, are expecting him to not have been "struck down" so much as ["made more powerful than you can possibly imagine.".
Old Captain Tunar may have had a bad case of The Hunter syndrome, along with a strong penchant for a Lack of Empathy for his crew. But Tunar seemed to be a genuinely decent guy while around Lion-O. When he dies as he makes one final strike at the Ramlak, it's still sad.
If he hadn't gone out the way he did and lived, it would have made his lifetime of hunting and isolation even emptier. As it was, the man was a Determinator to the end.
The entirety of "Song of the Petalars":
The adorable baby Petalar being welcomed into the world, the wizened elder dissolving into wind-blown leaves the next.
The baby could be Emrick. Wonder if that means the elder was Emrick's father or grandfather?
"Yes, my friend. Many adventures I hope." Lion-O had learned the Petalars live for only one day. He's got this bittersweet grin and sad realization in his voice that just...(sniff)
Emrick's Rousing Speech to the peaceful Petalars: "Let this day be sung about by our people for ages to come!" Remembering, of course, that a Petalar lifetime lasts roughly a day. And then his death and funeral, in Lion-O's hands, with no regrets.. And the Petalars' singing. And the kittens cry! And then they hit us with a Really Dead Montage as well.
That's nothing, Lion-Ocries!
In "Journey to the Tower of Omens." To save Lion-O, Jaga's secondHeroic Sacrifice. It's the way Cheetara cradles the shattered remains of his lantern that really drives it home.
In "Berbils" Ro-Bear Bill after he was attacked trying to save his family...Poor guy.
In "Into the Astral Plane"
Little Cheetara begging to join the Clerics after failing the test is heartbreaking. She makes it clear that she has no place to go and no family to turn to, so joining the Clerics was her chance to get out of poverty.
Cheetara showing patience by waiting outside the Cleric's building; Tygra giving her a flower and hoping that she'd notice him. While Cheetara's patience eventually paid off...Tygra's hasn't. Turns out he's not a Jerk with a Heart of Jerk, he's really a Jerk with a Heart of Gold, and this troper hopes that he's going to be wrong about Cheetara never 'seeing' him, whatever happens with shipping.
Tygra's patience was finally paid off in "Between Brothers". Cheetara told Tygra she chose him after he gave her the flower, which encourage her to not give up. Unfortunately, Lion-O saw their first kiss.
In "Trials of Lion-O, Part 2" when Lion-O fails the final trial. The way Trial!Tygra says "I'm sorry" as he hands him the death-key is heartbreaking.
Kit out right refusing to believe that Lion-O is dead is this or heartwarming.
How absolutely heartbroken each of the others sounds while trying to convince her otherwise doesn't help matters.
The farewell between Tygra and his father. And his entire clan.
The whole of "Native Son", in fact, particularly the end where we learn that the tiger clan has been Dead All Along and kept on earth by the curse, and with it broken, they go into the light - leaving Tygra the last of his clan.
The scene where Tygra's father is trying to convince him to leave. He's doing it for Tygra's safety and wellbeing, as practically the entire clan wants to kill him in order to break the curse. However, knowing that Tygra wouldn't accept that, he instead insults and belittles him, telling him that living among lions has made him soft, and that he can never truly consider him a tiger, let alone a son. It's all an act, of course, but Tygra buys it completely, and his father's heartbreak at having him leave again is evident. What really sells it, though? Tygra's "I can't believe I came this close to forgiving you".
A happier one towards the end. After the Tiger clan depart into the light, Tygra angrily claims "Am I destined to lose everything I care about?". Lion-O responds "You'll never lose me, brother". To which Tygra responds with...a happy smile. He DOES care!
"Survivial of the Fittest" was essentially a Breather Episode after the previous one, but even then we still got the Thunderkittens losing their father and running away to reduce the strain on their mother and siblings.
In the same episode, it shows their early days in the slums of Thundera, before they turned to thievery for survival. They tried to live honestly, begging for scraps and spare coins, only to be ignored by passers-by. And when someone drops his coin purse without noticing, they resist the temptation to take it, only to be accused of theft when they try to return it.
Pumyra's "The Reason You Suck" Speech to Lion-O is heartbreaking, and towards the end of it she almost cries herself. It's the first sign that under all of her anger there is something more. She honestly believed in Lion-O (not something many cats would claim) and was devastated when he never came.
Oh, and to twist the knife even further, that whole reconciliation moment she had with him by the end of that same episode was all an act. She was an all too eager agent of Mumm-Ra's the whole time. And the flashback explaining it is just plain heartbreaking.
The ending to "The Soul Sever". The Soul Sever loses the souls of his wife and children, despite dedicating most of his life to saving them, reducing him (mind you, he's a 'robot' at this point) to tears. By the very end of the episode he's left alone in his broken down base, holding the tiny broken down body of an insectoid robot. To make things worse, the way his family leaves is rather unceremonious too. They don't re-materialize to give their last goodbyes, or even say whether they really wanted to pass on at all, they just float up into the sky while The Soul Sever cries out in anguish after them.
The series cancellation is this. Despite gathering heavy praise from critics and fans for its storylines and characters, Cartoon Network kept moving its time slot and did little to advertise it and before the Season Finale got cancelled despite the protests and efforts of its fanbase and those who worked on it to keep it on. Toonami picked it up and aired it on its Midnight Run and the show got good ratings considering its timeslot, matching over a million viewers and at times beating their flagship shows. Now that its run its episode course, its been taken off the air. Damn it Cartoon Network.