The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP−/− mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP−/− mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP−/− and wt mice were infected with the murine γ-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-γ was significantly higher in SAP−/− mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP−/− mice, which indicates that SAP−/− CTL control this infection more efficiently than wt CTL. Finally, we found that the Vβ4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP−/− mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.