Hematopoietic stem and progenitor cells (HSPCs) arise during embryonic development and are essential for sustaining the blood and immune systems throughout life. Tight regulation of HSPC numbers is critical for hematopoietic homeostasis. Here, we identified DEAD-box helicase 41 (Ddx41) as a gatekeeper of HSPC production. Using zebrafish ddx41 mutants, we unveiled a critical role for this helicase in regulating HSPC production at the endothelial-to-hematopoietic transition. We determined that Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular fitness. Excess R-loop levels in ddx41 mutants triggered the cGAS-STING inflammatory pathway leading to increased numbers of hemogenic endothelium and HSPCs. Elevated R-loop accumulation and inflammatory signaling were observed in human cells with decreased DDX41, suggesting possible conservation of mechanism. These findings delineate that precise regulation of R-loop levels during development is critical for limiting cGAS-STING activity and HSPC numbers.