Many viruses, including Herpes Simplex Virus (HSV), have developed strategies to avoid detection by cytotoxic T lymphocytes (CTLs). In this article, we evaluated the role of individual HSV-1 genes in preventing cytolysis and apoptosis, and in decreasing viral yield after CTL exposure of HSV-infected fibroblasts, using viruses deleted for the immune evasion gene Us12 or one of the two antiapoptotic genes Us3 and Us5. To evaluate CTL-mediated apoptosis, we used a flow cytometry assay measuring active caspase-3 in target cells. This assay was more sensitive than the chromium release assay used to evaluate cytolysis, and measured a different aspect of CTL cytotoxicity. Although virus with deletion of Us12 was markedly defective in the ability to prevent lysis of target fibroblasts, it retained most of its ability to protect target fibroblasts from CTL-induced apoptosis. Virus with deletion of Us3 was also defective in the ability to prevent lysis of target fibroblasts, yet such virus protected target fibroblasts from CTL-induced apoptosis as well as wild-type viruses. In contrast, Us5-deleted virus showed defects in the ability to protect target fibroblasts from both cytolysis and apoptosis after CTL attack. In addition, the replication of Us12-deleted virus was reduced compared with wild-type virus in fibroblasts subjected to CTL attack 6 h after infection, but showed equivalent replication when CTL attack occurred later. In contrast, Us3- or Us5-deleted virus showed no measurable defect in their ability to replicate in fibroblasts under CTL attack. Our data suggest that cytolysis, apoptosis, and viral yield do not necessarily correlate in infected cells under CTL attack. Furthermore, the Us3, Us5, and Us12 viral genes each have unique inhibitory effects on the different T lymphocyte cytotoxic effects. Taken together, these results suggest that HSV evasion of cellular immunity is multifacterial and complex, and relies on the partially redundant activities of various individual HSV proteins.