The two-signal theory of T-cell activation dictates that optimal T-cell responses are determined by a least two signals, the primary signal provided by the antigen-receptor complex (TCR/CD3) and the second signal provided by a costimulatory receptor. Recent studies have underlined the importance of in trans costimulation via CD28 in the regulation of transplant rejection. Previous studies have emphasized the ability of CD28 to operate in cis in the amplification of signaling through the T-cell receptor (TCR). Our recent work has demonstrated that CD28 can activate the lipid kinase phosphatidylinositol 3-kinase (PI-3K) and can cooperate with adapters Vav and SLP-76 to influence the induction of interleukin (IL)-2 and IL-4 transcription in the absence of TCR ligation. CD28-PI-3K binding and CD28-VAV/SLP-76 cooperativity provide a pathway to account for in trans costimulation in T-cell immunity.