The acquisition of immunologic self-tolerance is governed, in part, by selection mechanisms that occur during intrathymic T cell ontogeny. Although considerable data exist for the molecular basis of mature T cell signal transduction, the enzymes that participate in thymic TCR selection processes have remained unidentified. We report that augmented thymic expression of the CD45R0 protein tyrosine phosphatase increased the efficacy of TCR-mediated apoptosis and MHC-restricted negative selection of HY TCRs in vivo. Additionally, augmented CD45R0 expression resulted in the activation of endogenous p56lck tyrosine kinase in CD4+CD8+ thymocytes. These results identify a cellular enzyme, the CD45R0 protein tyrosine phosphatase, involved in the regulation of apoptosis and TCR selection mechanisms during CD4+CD8+ thymocyte differentiation.