TLRs play a critical role in inducing inflammatory and immune responses against microbial agents. In this study, we have investigated the role of NF-κB transcription factors in regulating TLR-induced gene expression in dendritic cells, a key APC type. The p50 and cRel NF-κB subunits were found to be crucial for regulating genes important for dendritic cell-induced T cell responses (eg, CD40, IL-12, and IL-18) but not for genes encoding inflammatory cytokines (eg, TNF-α, IL-1α, and IL-6). In striking contrast, the RelA subunit was crucial for expression of inflammatory cytokine genes but not T cell stimulatory genes. These novel findings reveal a fundamentally important difference in biological function of genes regulated by different NF-κB subunits. Focusing on RelA target gene specificity mechanisms, we investigated whether the κB site and/or the unique composition of RelA played the most crucial role. Surprisingly, studies of IL-6 expression showed that the κB site is not a primary determinant of RelA target gene specificity. Instead, a major specificity mechanism is the unique ability of RelA to interact with the transcriptional coactivator CREB-binding protein, a function not shared with the closely related cRel subunit. Together, our findings indicate novel and critically important overall roles of NF-κB in TLR-induced gene expression that are mediated by unique functions of distinct subunits.