A continuing concern with CD28 and/or CD40 blockade-based strategies to induce tolerance and mixed chimerism is their potential to disrupt protective immunity to preexisting infections. In this report, we find that preexisting persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13 prevents the induction of tolerance, mixed chimerism, and donor-reactive T cell deletion. Mice continue to be refractory to tolerance induction even after viremia has been resolved and virus is present only at very low levels in peripheral tissues. Conversely, we find that the full tolerance regimen, or costimulation blockade alone, specifically inhibits already ongoing antiviral immune responses, leading to an inability to control viremia. These findings suggest that ongoing T cell responses continue to depend on costimulatory interactions in the setting of a chronic infection and provide insight into potential risks following costimulation blockade posed by chronic or latent viral infections such as hepatitis C, EBV, and CMV.