Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45⁺ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.