Yellow fever virus (YFV) is a reemerging problem despite the existence of an effective live-attenuated vaccine. The induction of YFV-neutralizing antibodies undoubtedly contributes to vaccine efficacy, but T lymphocyte responses to YFV likely play a role in long-term efficacy. We studied the T lymphocyte responses to YFV in four vaccinees. Proliferation and cytolytic responses to YFV were demonstrated in all subjects. We isolated 13 YFV-specific CD8⁺ CTL lines that recognized epitopes on the E, NS1, NS2b, and NS3 proteins; eight CTL lines were HLA-B35-restricted. YFV-specific T cell responses were detectable by IFNγ ELISPOT assays 14 days postvaccination, with T cell frequencies sustained for up to 19 months. To our knowledge, this is the first report of human T lymphocyte responses following YFV vaccination. These results indicate that the live 17D YFV vaccine induced CD8⁺ T cell responses directed against at least four different HLA-B35-restricted YFV epitopes.