Previous studies have suggested that, differing from model Ags, viruses that replicate extensively in the host still induce normal CD8+ T cell responses in the absence of CD28 costimulation. Because these studies were performed with viruses that do not normally cause acute disease, an important remaining question is whether CD28 costimulation is required for CD8+ T cell-mediated resistance to widely replicating but pathogenic viruses. To address this question, we studied the role of CD28 costimulation in CD8+ T cell-mediated resistance to mousepox, a disease of the mouse caused by the natural mouse pathogen, the ectromelia virus (ECTV). C57BL/6 (B6) mice are naturally resistant to mousepox, partly due to a fast and strong CD8+ T cell response. We found that B6 mice deficient in CD28 (CD28 knockout (KO)) are highly susceptible to lethal mousepox during the early stages of ECTV infection but can be protected by immunization with the antigenically related vaccinia virus (VACV) or by adoptive transfer of CD28 KO anti-VACV memory CD8+ cells. Of interest, a thorough comparison of the CD8+ T cell responses to ECTV and VACV suggests that the main reason for the susceptibility of CD28 KO mice to mousepox is a reduced response at the early stages of infection. Thus, while in the absence of CD28 costimulation the end point strength of the T cell responses to nonpathogenic viruses may appear normal, CD28 costimulation increases the speed of the T cell response and is essential for resistance to a life-threatening acute viral disease.