Abstract Body: ETV2 is a master regulator and pioneer factor for the endothelial lineage. Previous studies have established that this transcription factor was essential for embryogenesis and direct reprogramming of fibroblasts to endothelial cells. ETV2-mediated direct reprogramming holds therapeutic potential in treating ischemic tissues including ischemic heart disease, yet the underlying molecular mechanisms remain to be completely defined. In the current studies, we focused on deciphering the mechanisms that governed ETV2-mediated reprogramming, including the role of RIG1 inflammatory responses. We hypothesized that ETV2 transcriptionally activated Rig1 gene expression, increased expression of its downstream network [RIG1-like receptor (RLR) pathway] and modulated the reprogramming of fibroblasts to endothelial cells. Mouse embryonic fibroblasts (MEFs) with inducible ETV2 expression system were used to overexpress ETV2, and single-cell RNA-seq defined transcript expression involved in RLR signaling pathways. ChIP-seq, electromobility gel shift assays (EMSA), and transcriptional assays were used to verify that ETV2 was a direct upstream activator of Rig1 gene expression. Both RIG1 and RLR pathway proteins were significantly upregulated in ETV2-overexpressing MEFs. ETV2 mediated the Rig1 upregulation through direct binding to the Rig1 promoter and transcriptional activation of Rig1 expression. Knockdown of Rig1 using shRNA significantly (p < 0.05) reduced the efficiency of fibroblast to endothelial cell reprogramming more than 70%. These results highlight that 1) ETV2 activates inflammatory pathways and 2) RIG1 is direct downstream target of ETV2 and together they have an essential role in the reprogramming process. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of a revascularization strategy for the treatment of ischemic heart disease.