Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSPCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized collection of endothelial cells (ECs) within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that cysteine rich intestinal protein 2 (crip2), a LIM domain-binding protein expressed specifically in the dorsal aorta, has a previously unrecognized function in establishing the proper EC environment for HSPC specification. To investigate the requirement of crip2 in HSPC development, we employed CRISPR mutagenesis to generate loss-of-function alleles in crip2 and crip3, a gene family member with expression in cardiovascular tissues, to avoid potential for genetic compensation. crip2-/-;crip3-/- (cripDM) embryos exhibit decreased emergence of HSPCs by 26 hours post fertilization (hpf) with impaired lineage derivative production. To decipher the mechanisms underlying the cripDM phenotype, we performed single cell RNA sequencing of sorted, kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and a failure to repress Notch signals in cripDM hemogenic endothelium during the vital transition of HE specification to HSPC emergence. Moreover, inhibition of Notch activation partially rescues the diminished HSPCs in cripDM embryos by facilitating HSPC generation. Through identification of Crip2 as a novel upstream regulator of Notch repression in HE, we anticipate that our insights will contribute to further understanding of the complex cues necessary to improve human HSC production in vitro.