CD28 provides an important costimulatory signal in T cell activation that regulates multiple cellular processes including proliferation and survival. Several signal transduction pathways are activated by CD28; however, the precise biochemical mechanism by which CD28 regulates T cell function remains controversial. Retroviral gene transfer into primary T cells from TCR-transgenic, CD28-deficient mice was used to determine the specific sequences within CD28 that determine function. Discrete regions of the cytoplasmic domain of CD28 were identified that differentially regulate T cell proliferation and induction of the anti-apoptotic protein Bcl-X L. Mutation of C-terminal proline residues abrogated the proliferative and cytokine regulatory features of CD28 costimulation while preserving Bcl-X L induction. Conversely, mutation of residues important in phosphatidylinositol 3-kinase activation partially inhibited proliferation but prevented induction of Bcl-X L. Thus the ability of CD28 to regulate proliferation and induction of Bcl-X L map to distinct motifs, suggesting independent signaling cascades modulate these biologic effects.