If I have teen titans and JLI team up, have roy harper arsenal, general glory, and robin team atk someone, I use teen titan go to ready roy harper and use his abilty to stun the defender, does that defender gets koed via general glory's effect : when 3 or more jli team atker stun a defender ko that defender?

No because he say's when three or more JLI team attackers stun a defender KO it. Since your Using Roy Harper's effect he's being stunned by his effect and not the team attackers.

More precisely, the defender is being stunned by 1 team attacker. Since he isn't being stunned by 3 or more, the effect doesn't rigger.

What about Play-room? Let's say the same thing above but replace General glory with an Avenger. I flip up play-room and use Roy's ability. Do they still lose 4 Endurance?

I'd assume so, because playroom says 1 or more, and roy would be a team attacker.

Would play-room work with Black Widow? Black Widows text:
While Natasha Romanoff is team attacking, whenever she stuns a defender, you may stun target character with cost 3 or less.

I would say no because After you stun someone you're no longer team attacking, right? This works the samw way Sinestro does in that the stun happens after the attack, right?

Playroom would trigger for Roy Harper in that situation, yes; he's an Avengers team attacker, and he stunned a defender.

When Black Widow's triggered effect stuns a character, not only is she no longer team attacking, but the character that she stunned wouldn't have been a defender. Playroom doesn't trigger. And, yes, the same rule applies here as applies to Sinestro; if an effect is triggered during attack conclusion, it will be placed on the chain after attack conclusion.