Stoke put faith in their ability to win their second appeal by signing him in the January transfer window and then loaning him out to Dutch club FC Utrecht.
Agudelo has since enjoyed moderate success in the Dutch Eredivisie by netting three times in eight starts and six sub appearances. He is currently waiting to see if he will be picked by Jurgen Klinsmann to join Stoke team-mate Geoff Cameron in the US World Cup squad.
Hughes says Agudelo has taken the news badly, telling The Sentinel: "We are desperately disappointed for the boy himself because he's made a big effort.
"He came to us and then went out to Holland because he wanted to do everything in his power to help himself."
Hughes says a third appeal is now a long shot as Stoke weigh up what to do with a player they are not allowed to play.
"Unfortunately, that's probably the end of it," Hughes reluctantly admitted.
"You never say never, but we would probably have to wait something like two years for him to play the required number of games."
For the big stories and the banter follow us on Twitter: @tribalfootball