NK cells play essential roles in eliminating virally infected cells and tumor cells. Polyinosinic-polycytidylic acid (poly I:C), a double-stranded RNA analog recognized by melanoma-differentiation associated gene 5 (MDA5) and TLR3, activates NK cells in vivo. MDA5 and TLR3 signal through distinct adaptor molecules, IFN-promoter stimulator-1 (IPS-1) and Toll/IL-1R domaincontaining adaptor inducing IFN-β (TRIF), respectively. However, it remains unclear how NK cells are activated by poly I:C in vivo. In this study, we demonstrate that the IPS-1-dependent and the TRIF-dependent pathways are essential for NK cell activation to poly I:C stimulation in mice, whereas deficiency in either IPS-1 or TRIF only modestly impairs the poly I:C-induced NK cell activation. Furthermore, both IPS-1 and TRIF contributed to suppression of implanted B16 tumor growth in response to poly I:C administration via NK cell activation. Presence of IPS-1 and TRIF in dendritic cells (DCs), but not NK cells, was required for production of IFN-γ to poly I:C in NK cells in vitro. Moreover CD8α+ conventional dendritic cells (cDCs), but not CD8α- cDCs, expressed genes for type I IFNs, IL-6, and IL-12p40 in response to poly I:C stimulation, and were also responsible for inducing IFN-γ production in NK cells. Taken together, poly I:C activates the IPS-1- and TRIF-dependent pathways in CD8α+ cDCs, which in turn leads to NK cell activation. Copyright © 2009 by The American Association of Immunologists, Inc.