Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called β5t. β5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of β5t into proteasomes in place of β5 or β5i selectively reduces this activity. We also found that β5t-deficient mice displayed defective development of CD8⁺ T cells in the thymus. Our results suggest a key role for β5t in generating the MHC class I–restricted CD8⁺ T cell repertoire during thymic selection.