Conjugation of TLR agonists to protein or peptide antigens has been demonstrated in many studies to be an effective vaccine formula in inducing cellular immunity. However, the molecular and cellular mediators involved in TLR-induced immune responses have not been carefully examined. In this study, we identify Type I IFN and IL-12 as critical mediators of cross-priming induced by a TLR7 agonist-antigen conjugate. We demonstrate that TLR7-driven cross-priming requires both Type I IFN and IL-12. Signaling through the IFN-αβR was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Although IL-12 was indispensable during cross-priming, it did not regulate DC function. Therefore, the codependency for these 2 cytokines during TLR7-induced cross-priming is the result of their divergent effects on different cell-types. Furthermore, although dermal and CD8α⁺ DCs were able to cross-prime CD8⁺ T cells, Langerhans cells were unexpectedly found to potently cross-present antigen and support CD8⁺ T-cell expansion, both in vitro and in vivo. Collectively, the data show that a TLR7 agonist-antigen conjugate elicits CD8⁺ T-cell responses by the coordinated recruitment and activation of both tissue-derived and lymphoid organ-resident DC subsets through a Type I IFN and IL-12 codependent mechanism.