Tumor necrosis factor-α (TNF-α) is well recognized for its role in mediating innate immune responses. However, the mechanisms of TNF-α that influence the adaptive immune response to virus infections are not well understood. In this study, we have investigated the role of TNF-α in activating the cellular and humoral responses to systemic viral challenge with recombinant replication-defective adenovirus (rAd). Evaluation of T cell function in TNF-α-deficient (TNFKO) mice revealed impaired virus-specific proliferation of T cells derived from the draining lymph nodes of the liver. Analysis of dendritic cells (DC) isolated from local draining lymph nodes after systemic challenge showed that DC from TNFKO mice were relatively immature compared with those from strain-matched wild-type mice. In vitro, TNF-α was required to mature DC efficiently during virus-mediated stimulation. Adoptive transfer of primed, mature DC into TNFKO mice restored T cell responses and reconstituted anti-adenovirus antibody responses. Thus, TNF-α plays a significant role in the maturation of DC after adenovirus challenge both in vitro and in vivo, highlighting the importance of this innate cytokine in activating adaptive immunity to viral challenge.