Abstract Engagement of the TCR and CD28 coreceptor by their respective ligands activates signal transduction cascades that ultimately lead to the activation of the transcription factors NFAT, AP-1, and NF-κB, which are required for the expression of cytokines and T cell clonal expansion. Previous studies have demonstrated that in mature T cells, activation of AP-1 and NF-κB is dependent on protein kinase C θ, suggesting the existence of a common signaling pathway. In this study, we show that in human primary CD4+ T cells, exposure to the cell-permeable IKKβ inhibitor PS-1145 or genetic ablation of IKKβ abrogates cell proliferation and impairs the activation of NF-κB and AP-1 transcription factors in response to engagement of CD3 and CD28 coreceptor. In addition, we show that stimulation of T cells in the absence of IKKβ activity promotes the time-dependent and cyclosporine-sensitive expression of negative regulators of T cell signaling leading to a hyporesponsive state of T cells.