Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation. Display omitted •Rig-I or Mda5 deficiency impairs inflammatory signaling and HSPC formation•Lgp2 deficiency enhances inflammatory signaling and HSPC formation•Overexpression of sine3-1a enhances HSPC formation by engaging Rig-I and Mda5•RLR deficiency in murine embryonic HSPCs recapitulates the phenotypes in zebrafish Inflammatory signaling is a key regulator of developmental hematopoiesis, but the mechanisms driving such signals are unknown. Lefkopoulos et al. now provide insight into the underlying mechanisms by revealing endogenous repetitive element RNAs as activators of RIG-I-like receptors during embryonic hematopoiesis. RIG-I-like receptor activation induces inflammatory signals necessary for hematopoietic stem and progenitor cell generation.