AbstractPoly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3−/− mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3−/− mice. A higher percentage of Ag-specific CD8 T cells became IFN-γ and TNF-α producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-α/β-dependent. Administration of IFN-α/β-neutralizing Abs abolished the poly(I:C) effects in TLR3−/− mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.