A pivotal obstacle to cancer immunotherapy is peripheral T cell tolerance to tumor-associated antigens (TAAs). Tolerance induction among mature T cells in the periphery operates through a variety of mechanisms, including anergy and apoptosis. Although Fas-FasL-mediated apoptosis is a well-defined tolerance inducing mechanism, direct evidence of its interference with TAA-specific immunity in vivo is still lacking. In this report, we used the TRAMP mouse, which expresses SV40 large T antigen (Tag) preferentially in the prostate and develops prostate tumors, as a model system to address the role of Fas-mediated apoptosis in regulating peripheral T cell tolerance. Using RT-PCR and tetramer staining to quantify TAA-specific TCR-expressing cytolytic T lymphocytes (CTLs), we have shown the presence of TAA-specific CTLs at higher levels in TRAMP mice than in syngeneic C57Bl/6 mice. Tag-specific immunization led to the expansion of Tag-specific CTLs in C57Bl/6 mice, and to their elimination in TRAMP mice. Interestingly, in TRAMP mice with deficient Fas (Hybrid TRAMP-lpr/lpr), Tag-specific CTL elimination in response to Tag immunization did not take place. The results of cytolytic-function assays were consistent with induction and elimination patterns of TAA-specific CTLs and those of RT-PCR and tetramer staining. In conclusion, our data show that Fas-mediated TAA-specific CTL apoptosis contributes to T cell tolerance and suggest that such tolerance could be potentiated following TAA-specific immunization.