Phosphatidylinositol 3(PI3)-kinase is implicated in various biological responses, including protection from apoptosis, although its role in antigen-induced T cell death and the molecular effectors it triggers remains ill-defined. Here, we investigated the role of PI3-kinase activity in the prevention of T cell receptor/CD3-induced cell death by CD28. PI3-kinase inhibitors blocked the up-regulation of Bcl-X(L) by CD28, without impairing the prevention of T cell receptor/CD3-triggered apoptosis by CD28, hence showing the existence of a cell-survival pathway independent of PI3-kinase activity and up-regulation of Bcl-X(L). Instead, we show that up-regulation of FasL which is instrumental in CD3-induced apoptosis was prevented upon CD28 co-stimulation. These results indicate that PI3-kinase couples CD28 to Bcl-X(L) up-regulation and provide a molecular basis for the role of CD28 in cell survival through a PI3-kinase-independent mechanism including FasL down-regulation.