Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) are double-stranded RNA (dsRNA)-recognizing receptors that mediate innate immune responses to virus infection. However, the roles played by these receptors in the pathogenesis of avian viruses are poorly understood. In this study, we generated TLR3 and MDA5 single knockout (SKO) and TLR3-MDA5 double knockout (DKO) quail fibroblast cells and examined dsRNA receptor-mediated innate immune responses in vitro. The knockout cells were then stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or influenza A virus. Endosomal stimulation of TLR3 by adding poly(I:C) in cell culture media or cytoplasmic stimulation of MDA5 by transfecting poly(I:C) resulted in significant increases of TLR3, MDA5, interferon (IFN) β, and interleukin 8 gene expression levels in wild type (WT) cells. Endosomal poly(I:C) treatment induced a higher level expression of most of the genes tested in MDA5 SKO cells compared with WT cells, but not in TLR3 SKO and DKO cells. Cytoplasmic transfection of poly(I:C) led to significant upregulation of all four genes in WT, TLR3 SKO, and MDA5 SKO cells at 8 hr posttransfection and negligible gene expression changes in TLR3-MDA5 DKO cells. Upon infection with a strain of influenza virus with compromised IFN antagonistic capability, WT cells produced the highest amount of biologically active type I IFN followed by TLR3 SKO and MDA5 SKO cells. DKO cells did not produce detectable amounts of type I IFN. However, the IFN did not induce an antiviral state fast enough to block virus replication, even in WT cells under the experimental conditions employed. In summary, our data demonstrate that TLR3 and MDA5 are the key functional dsRNA receptors in quail and imply their coordinated roles in the induction of innate immune responses upon virus infection.