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Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3−/−×Irf7−/− double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3−/−×Irf5−/−×Irf7−/− triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar−/−). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar−/− mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs−/− mDC. The relative equivalence of TKO and Mavs−/− responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5., Author Summary Host pathogen sensors, including those of the Toll-like receptor and RIG-I like receptor (RLR) families, detect viral infection in cells. Signaling through these receptors triggers expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), in part through the IRF family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF-3 and IRF-7 control IFN expression in fibroblasts and neurons, whereas macrophages and myeloid dendritic cells (mDC) retained the ability to induce IFN-β without IRF-3 and IRF-7. In the current study, we generated Irf3−/−×Irf5−/−×Irf7−/− (TKO) mice to characterize the contributions of specific IRF transcription factors to IFN and ISG induction in response to WNV infection in cells and in mice. We found that induction of IFN and ISGs was largely abolished in TKO mDC, but sustained in TKO macrophages. Because IFN and ISG induction also was absent in mDC lacking MAVS, a key mediator of RLR signaling, our results suggest a novel signaling link between IRF-5 and MAVS. This study establishes the molecular pathways responsible for IFN induction in mDC and suggests a cross-talk between IRF-5 and RLR signaling pathways.