Binding sites for the nuclear factor (NF)-κB transcription factor have been identified within control regions of many genes involved in inflammatory and immune responses. Such κB sites are often found adjacent to those of interferon (IFN)-γ–inducible transcription factors, suggesting a requirement for multiple signaling pathways for gene regulation. Using fibroblasts from RelA (p65)-deficient mice generated by gene targeting, we have investigated the role of this subunit of NF-κB in gene activation by microbial lipopolysaccharide, tumor necrosis factor α, and in possible synergism with the IFN-γ–signaling pathway. Our results indicate not only that RelA is required for activation of key genes involved in adaptive (acquired) immune responses, including major histocompatibility complex class I, CD40, and the Fas death receptor, but also that both NF-κB–inducing signals and IFN-γ are necessary for maximal activation. In contrast, neutrophil-specific chemokine genes KC and MIP-2, which can function as nonspecific mediators in innate immune responses, were strongly induced by RelA in the absence of IFN-γ. Our results show that RelA plays a critical role in activation of immune system genes in response to nonspecific stimuli and demonstrate a novel proapoptotic function for this protein in Fas-induced cell death.