The immune response to embryonic stem (ES) cells is still poorly understood. In this study, we addressed the adaptive cellular immune response to undifferentiated and differentiated ES cells infected with lymphocytic choriomeningitis virus (LCMV), a vertically transmitted pathogen in mice and humans. In contrast to the prevailing view, we found that undifferentiated and differentiated murine ES cells express MHC class I molecules, although at low levels. When cocultured with LCMV-infected ES cells, syngeneic but not allogeneic LCMV-specific CTL secrete IFN-gamma. Strikingly, LCMV-specific CTL do not efficiently kill LCMV-infected ES cells. ES cells showed high-level expression of the serine protease inhibitor 6, an endogenous inhibitor of the CTL-derived cytotoxic effector molecule granzyme B. Down-regulation of serpin-6 by RNA interference sensitized ES cells for CTL-induced cell death. The results of this study suggest that LCMV-infected murine ES cells present viral Ags and are recognized by LCMV-specific CTL in a MHC class I-restricted manner, yet resist CTL-mediated lysis through high-level expression of serine protease inhibitor 6.