Trixie's horrified and confused reaction to seeing her undead body for the first time.
"What am I?"
Despite Twilight's best efforts, Trixie is fully aware about the ponies who had just been liberated from her fiasco with the Alicorn Amulet looking at her with fear and contempt.
Not to mention the cruel irony that a pony who loves the attention of others, can no longer walk around without a cloak.
Applejack driving Trixie off, after she saw her talking to Applebloom. Made especially heart breaking, because neither one of them meant any harm. (It was Applebloom that started talking to Trixie and Applejack was just jumping in to protect her sister.)
Trixie's second death and subsequent funeral, is a very somber and heartbreaking moment, that leaves the entire cast with Heroic BSoD.
Not helped by how sudden it was, as the last chapter being a fairly heartwarming and mellow breather episode, up until the very end.
Pinkie gets hit especially hard, after it is revealed that the rock farm "Trixie didn't deserve to work on" was her families’ and they lost more than just a friend.
The Grim Reaper of Travelers and Trixie’s long lost mother has her chance to finally reunite with and let her daughter rest in peace, ripped from her by a force no one knew about.
