To demonstrate the importance of DHX29 expression, several studies have shown that DHX29 is essentially required for protein translation, cell proliferation and survival [31–33, 44]. Consistent with these results, we show that KD or KO of DHX29 leads to cell death after 5 days in cell culture. More importantly, we demonstrate that both DHX29 and MDA5 function as RNA co-sensors and are required for the dsRNA-binding and type I IFN signaling in response to HMW poly I:C and EMCV, but not VSV infection. Our data on the specific recognition of EMCV by DHX29 are quite concordant with several recent studies showing the absolute requirement of DHX29 for the formation and scanning of 43S translation initiation complexes on mRNAs with 5´-structured UTRs, such as those from EMCV [31–33]. The translation of such mRNAs requires DHX29 in 43S translation initiation complexes to unwind structured mRNA and identify the correct initial start codon [34, 35]. These studies also suggest that DHX29 can recognize mRNAs with 5´-structured UTRs present in many Picornaviridae family viruses, including EMCV. Our results obtained with shRNA-mediated KD as well as CRISPR/Cas9-mediated KO of DHX29 in various cell types, further substantially demonstrate the specificity and requirement of DHX29 as an RNA co-sensor in the innate immune response to HMW Poly(I:C) treatment and EMCV infection. Moreover, the presence of DXH29 in both human and mouse cells supports for its functional conservation between human and mouse. Hence, our results unequivocally establish that DHX29 is expressed in all types of immune cells tested, including PBMC, monocyte, B cells, T cells, macrophages and BMDC, and is essentially required for cell proliferation and survival; Importantly, our results have clearly demonstrated that DHX29 functions as an MDA5-specific RNA sensor in response to HMW Poly(I:C) and EMCV, but not to LMW Poly(I:C) or VSV, in immune cells tested. Further evidence shows that DHX29 alone cannot trigger type I IFN signaling in immune cells; it must interact with MDA5, but not RIG-I, to activate the type I IFN signaling pathway in various immune cell types, including THP-1, human PBMCs, murine macrophages and DCs. Of note, DHX29 does not interact with MDA5 in uninfected or untreated cells, but rather is induced after HMW Poly(I:C) treatment or EMCV infection. DHX29 does not interact with LGP2 or RIG-I even after viral infection, suggesting that the N-terminus of DHX29 specifically binds to the N-terminal (CARD-containing) domain of MDA5. However, unlike full-length DHX29, the truncated N-terminus of DHX29 (without the C-terminus) inhibits MDA5-mediated type I IFN signaling, suggesting that full-length DHX29 is required for its ability to enhance type I IFN signaling.