Hematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout life. They arise during embryogenesis from the hemogenic endothelium (HE) located in the floor of the main embryonic artery, the dorsal aorta. Our understanding of the mechanisms underlying HE specification remains incomplete, but precise regulation of the transcription factor Gata2 is crucial for the generation of HSCs. For example, CRISPR/Cas9-mediated deletion of a conserved intronic enhancer (i4 enhancer) leads to a decrease in gata2a expression and subsequent loss of HSCs. By contrast, knockdown of the Lim-domain-only protein Lmo4, previously identified as a binding partner of Ldb1, results in loss of HSCs and massively increased gata2a expression in the HE. HSC formation in lmo4a morphants can be rescued by inhibiting Gata2 activity. Thus, the dose of gata2a in HE is critical for de novo production of HSCs, and gata2a levels are controlled by a critical negative input from Lmo4. We have found that the zebrafish i4 enhancer drives GFP expression in endothelial cells, including the HE, and have established a transgenic zebrafish line to monitor its activity in vivo. This line provides high temporal resolution for monitoring changes in Gata2a activity in the hematopoietic lineage and we could detect increased i4 enhancer activity upon Lmo4 loss-of function. To unravel the molecular basis for the loss of HSCs upon altered Gata2a expression we have generated deletion mutants for the i4 enhancer and for lmo4a, in order to respectively lower and elevate the levels of Gata2a in the embryo. Our current focus includes transcriptome-wide gene expression analysis and genome-wide analysis of chromatin structure in both mutant backgrounds in endothelial cells, including the HE. This will determine the global impact on gene regulation caused by modulation of gata2a levels and will help us understand how the precise control of gata2 specifies the HE and generates HSCs.