Simultaneous blockade of the CD40 and CD28 T cell costimulatory pathways effectively promotes skin allograft survival in C3H/HeJ mice, extending median survival times (MSTs) beyond 100 days. This strategy is markedly less effective in C57BL/6 mice, with MSTs ranging between 20 and 30 days. In this study, we investigate the underlying genetic causes of these distinct phenotypes. Using H-2 congenic mice, we show that the genetic basis for the varied responses between these two strains is independent of the H-2 locus and T cell precursor frequency. C57BL/6 mice treated with costimulation blockade are able to generate allospecific CTL- and IFN-γ-producing T cells within 3-4 wk posttransplant, whereas mice with a C3H background generate neither CTL- nor IFN-γ-producing cells. Thus, differences appear to be in the generation of the immune response and not T cell homing. Strain differences in costimulation blockade-induced hyporesponsiveness persist in the absence of CD4+ T cells, implying a direct effect on CD8+ T cells. We demonstrate that genetic differences are important in cells of hemopoietic origin and that the costimulation blockade-resistant phenotype is dominant. Analysis of BXH recombinant inbred strains indicates that multiple loci contribute to the phenotype, and that the blockade resistance loci are preliminarily linked to 17 markers on four chromosomes. We conclude that strain variation in allograft MSTs following CD40/CD28 blockade results from the ability of CD8+ T cells in some strains to use alternative modes of costimulation to mount an effective alloresponse.