C57BL/6 (B6; H-2b) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2b congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2b spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2b cells function as “veto” cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2b veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8+ CTL were identified by a labeled Kb/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2b veto cell-mediated depletion of these CTL. A 65–93% reduction in the number of B6 Kb/KSPWFTTL tetramer+ CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2b veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2b veto cells) to block inhibition demonstrated that AKR.H-2b veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer+ CTL cocultured with AKR.H-2b veto cells was annexin V positive and Fashigh, indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2b cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vβ4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.