Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.