Thymocyte development requires positive selection of clones that can recognize Ag presented by MHC molecules and negative selection of clones that are self-reactive. However, the costimulatory signals required for negative selection and cell death, or positive selection and the transition to the peripheral lymphoid system, are not well understood. Many molecular interactions that are important for T cell activation have also been found to play a role in thymocyte development. The importance of the CD28/B7 interaction in the activation of mature T cells and recent observations that CD28 may play a role negative selection of developing CD4+CD8+ thymocytes suggest that CD28 may also be involved in development and maintenance of T cell tolerance. CD28-deficient mice were crossed to αβ and γδ TCR transgenic as well as H-2(k) Mls(c)-bearing animals and were used to address the role of CD28 in positive and negative selection of developing T cells. The CD28-deficient animals demonstrated no obvious deficiency in either positive or negative selection of developing thymocytes. However, when mixed bone marrow chimeras were created with cells derived from both CD28-deficient and wild-type mice, the CD28+ T cells had a selective advantage over the CD28-deficient T cells. Therefore, it appears that CD28, although not essential for the selection of T cells during development, may allow for additional signals that increase the efficiency of selection and/or expansion of peripheral T cell populations.