We explored the role of the NF-κB pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-κBα (inhibitor of κBα factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-x L features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-x L promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-κB site around− 840 decreased, while ectopic expression of I-κB kinase-β (IKKβ) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKKβ, interfered in the activation of the Bcl-x L promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-κB factors that bind to the NF-κB binding site in the Bcl-x L promoter, as well as the expression of Bcl-x L protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-κB activity, contained elevated levels of Bcl-x L protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-α. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-x L expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKKβ and expression of Bcl-x L promote the survival of primary human CD4+ T lymphocytes.