Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the Kb-binding S208–215 epitope 1; processing endogenous HBsAg generates theKb-binding S190–197 epitope 2. Cross-reactive CD8+ T cell responses were primed to epitope 1 but not epitope 2 when mice were immunized with natural HBsAgayw, orHBsAgadw2 variants differing within both epitopes by one or two residues. Expression of HBsAgayw from a transgene in the liver renders (HBs-tg) mice tolerant to epitope 1 of HBsAgayw. CD8+ T cells specific for epitope 1 could be primed in HBs-tg mice by HBsAgadw2; these specific CD8+ T cells cross-reacted with epitope 1 processed from the transgene-encoded HBsAgayw. The liver of vaccinated HBsAgayw transgenic mice showed severe histopathology and contained functional (IFNγ-producing), cross-reactive CD8+ T cells, and vaccinated HBs-tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross-reactive, specific CD8+ T cellimmunity that breaks tolerance to HBsAg.