Colombian Jairo Castillo and fellow forward Arnhold Rivas were suspended while midfielder Jorge Cardenas is training with the reserves following the club's decision after Queretaro lost 5-0 to Toluca in the Clausura championship at the weekend.
"The players need to know what we are facing: relegation, something very serious," club president Ulises Zurita said.
"We need cold heads and hot hearts but players who are not committed don't help us," he told reporters on Monday night.
Queretaro's Argentine-born Uruguayan coach Gustavo Matosas was given the club's backing with Zurita saying "for now he has our unconditional support".
Queretaro, bottom of Group Three with four points from six matches, meet Santos Laguna, runners-up in the Apertura in the first half of the season, on Saturday.
Puebla, also with four points, have signed Uruguayan former Indios coach Hector Eugui to take charge of their team having sacked Jose Luis Trejo. Puebla face Chiapas on Sunday.
Tigres UANL lead Group One with 13 points, three more than Santos Laguna. Toluca and San Luis are equal top of Group Two with 12 points and Pumas UNAM lead Group Three with 12, two more than title favourites Cruz Azul.
The top two in each of the three groups and the two next best-placed teams in the overall standings after the 17-match round robin phase qualify for the quarter-finals.
