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She starts using it as a greeting towards the other mane 5 in the EQG universe and they're all staring at her like "????????"
amusing thought: since Equestria is mostly full of creatures that don't have hands, they probably don't have the gesture of giving the middle finger, and wouldn't know what it means if someone did it to them
However, Discord, interdimentional chaos god, has both hands and would absolutely know what it signifies beyond our forth wall.
I.E.: Discord trying to stay good and not upset Fluttershy so when he's annoyed at someone he flips them off, because it makes him feel better, and they have no idea what it means anyway
Funnier thought: Twilight once again visits the human world, and, not knowing what it means (or being convinced it means something else), does it to someone and has to learn in the most humiliating way possible that he's been telling her to fuck off all this time