The cells that stimulate positive selection express specialized proteasome β-subunits different from those expressed by all other cells, including those involved in negative selection. Mice that lack all four specialized proteasome β-subunits, and therefore express only constitutive proteasomes in all cells, had a profound defect in the generation of CD8⁺ T cells. While a defect in positive selection would reflect an inability to generate the appropriate positively selecting peptides, a block at negative selection would point to the potential need to switch peptides between positive selection and negative selection to avoid the two processes' often cancelling each other out. We found that the block in T cell development occurred around the checkpoints of positive selection and, unexpectedly, negative selection as well.