CD28 has been demonstrated to play an important role in augmenting T cell proliferation and effector function. Costimulation through CD28 has also been reported to enhance human T cell survival. in this report, we have further investigated the role of CD28 in regulating T cell survival by comparing the survival characteristics of T cells from wild-type and CD28-deficient mice. CD28 costimulation of anti-CD3-activated cells augmented the viability of T cells from wild-type but not from CD28-deficient mice. CTLA4Ig treatment reduced wild-type T cell viability to a level comparable with CD28-deficient T cells. The ability of CD28 to enhance survival during T cell activation correlated positively with its ability to up-regulate the protein product of the cell survival gene bcl-xL. No differences in the expression of either Bcl-2 or Fas were observed between wild-type and CD28-deficient T cells. The CD28-dependent enhancement of cell survival during in vitro activation was found to be independent of Fas expression, as CD28 costimulation enhanced T cell survival to comparable levels in both wild-type and lpr animals. Cell death in CD28-deficient animals and in wild-type animals treated with CTLA4Ig displayed the morphologic characteristics of apoptosis. Additionally, inhibitors of ICE proteases could reverse cell death induced by TCR engagement in the absence of CD28 costimulation. Thus, CD28 costimulation not only enhances the proliferative expansion of cells activated through the TCR but also increases the likelihood that individual cells survive during T cell activation.