C57BL/6 mice mount a cytotoxic T-lymphocyte (CTL) response against the Daniel’s strain of Theiler’s murine encephalomyelitis virus (TMEV) 7 days after infection and do not develop persistent infection or the demyelinating syndrome similar to multiple sclerosis seen in susceptible mice. The TMEV capsid peptide VP2_121–130sensitizes H-2D ^b+ target cells for killing by central-nervous-system-infiltrating lymphocytes (CNS-ILs) isolated from C57BL/6 mice infected intracranially. D^b:VP2_121–130 peptide tetramers were used to stain CD8⁺ CNS-ILs, revealing that 50 to 63% of these cells bear receptors specific for VP2_121–130 presented in the context of D^b. No T cells bearing this specificity were found in the cervical lymph nodes or spleens of TMEV-infected mice. H-2^b mice lacking CD4, class II, gamma interferon, or CD28 expression are susceptible to persistent virus infection but surprisingly still generate high frequencies of CD8⁺, D^b:VP2_121–130-specific T cells. However, CD4-negative mice generate a lower frequency of D^b:VP2_121–130-specific T cells than do class II negative or normal H-2^b animals. Resistant tumor necrosis factor alpha receptor I knockout mice also generate a high frequency of CD8⁺ CNS-ILs specific for D^b:VP2_121–130. Furthermore, normally susceptible FVB mice that express a D^b transgene generate D^b:VP2_121–130-specific CD8⁺CNS-ILs at a frequency similar to that of C57BL/6 mice. These results demonstrate that VP2_121–130 presented in the context of D^b is an immunodominant epitope in TMEV infection and that the frequency of the VP2_121–130-specific CTLs appears to be independent of several key inflammatory mediators and genetic background but is regulated in part by the expression of CD4.