Toll-like receptors (TLRs) are able to interact with pathogen-derived products and their signals induce the coordinated activation of innate and adaptive immune mechanisms. Dendritic cells (DCs) play a central role in these events. As the different TLRs are able to trigger MyD88/TRIF-dependent and -independent signaling pathways, we wondered if the simultaneous activation of these signaling cascades would synergize with respect to DC activation and induce superior cytotoxic T-lymphocyte (CTL) activity in vivo. We observed that indeed the combined activation of MyD88-dependent and -independent signaling induced by TLR7 and TLR3 ligands provoked a more rapid and more sustained bone marrow–derived DC (BMDC) activation with regard to the secretion of proinflammatory cytokines, like IL-6 and IL-12p70, and the expression of costimulatory molecules like CD40, CD70, and CD86. Furthermore, in the presence of combined TLR ligand–stimulated DCs, CD4⁺ and CD8⁺ T cells were insensitive toward the inhibitory effects of regulatory T cells. Most importantly, peptide-loaded BMDCs stimulated by TLR ligand combinations resulted in a marked increase of CTL effector functions in wild-type mice in vivo. Thus, our results provide evidence that unlocking the full potential of DCs by advanced activation protocols will boost their immunogenic potential and improve DC-based vaccination strategies.