The US national team's numbers in the desert are now down to 20.
DaMarcus Beasley and Michael Orozco will not be available for Wednesday's friendly vs. Mexico, as Puebla have denied US Soccer's request for their release, the federation announced on Monday.
Head coach Jurgen Klinsmann will not add any replacements, reducing the roster to 20 players available for selection.
"We are very disappointed in the decision by Puebla," Klinsmann said in a statement. "When the match was scheduled, it was with the spirit that both teams would have access to their domestic-based players for what is a very important game for those competing for spots in the World Cup.
"Both MLS and most clubs in Liga MX honored that idea. While we understand their position, it's a huge opportunity lost for DaMarcus and Michael and doesn't contribute to the continuation of this respected rivalry."
Puebla are fighting for their top-flight survival in Mexico, currently languishing in last place in the Liga MX and just five points ahead of Atlante in the relegation standings with four games remaining. Beasley and Orozco, both starters, helped La Franja secure a 1-1 draw at home on Sunday against defending champions Club León.
The USMNT began gathering in Phoenix on Sunday ahead of Wednesday's friendly at University of Phoenix Stadium (11 pm ET, ESPN, UniMas).
