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Hematopoietic stem cells (HSCs) are at the foundation of the hematopoietic system. Endowed with multi-lineage differentiation and long-term self-renewal properties, HSCs have the capacity to replenish the entire blood system, thereby meeting the constant need for short-lived blood cell types throughout life. In all vertebrates, the first HSCs originate during embryonic life. Specialized hemogenic endothelial cells bud from the endothelial lining of the main arteries, such as the aorta, to undergo an endothelial-to-hematopoietic transition (EHT). EHT leads to the formation of hematopoietic cells that organize in clusters (intra-aortic hematopoietic clusters; IAHCs) that remain transiently attached to the endothelium. The number of IAHC cells peaks at E10.5, when the first HSCs start to be detected in the aorta. IAHCs contain very few HSCs and committed erythroid-myeloid progenitors, and are mainly composed of HSC precursors (pre-HSCs) that progressively mature towards HSCs. The decrease of IAHC cell number in the aorta coincides with the appearance of HSCs in the fetal liver (FL). Given the similar number of aortic pre-HSCs and HSCs in FL, pre-HSCs most likely migrate from the aorta to the FL to complete their maturation process there. This maturation, followed by expansion of the HSCs, leads to the formation of the adult HSC pool that colonizes the bone marrow (BM), the main site of hematopoiesis in adulthood. The restricted spatial and temporal production of HSCs during embryonic development is regulated by a complex network of cell-intrinsic and -extrinsic factors. Obtaining a better understanding of HSC regulation is the main focus of this thesis. The present work, begins to elucidate the complex regulatory network that controls HSC generation and pre-HSC maturation at different levels. We show that cell-intrinsic factors, such as molecular changes in the transcription factor network, allow for the transition from an endothelial to a hematopoietic program in the aorta. At the cellular level, complex rearrangements of cytoskeletal elements are needed for the successful emergence from the endothelium. The maintenance of the initiated stem cell program is critically dependent on the interaction of the cell with the surrounding microenvironment and its ability to respond to gradients of soluble factors. Impaired intra-cellular trafficking of receptors such as c-Kit, which mediates the cross-talk between HSCs and the niche factors, results in the loss of stem cell properties. Our results suggest that the appropriate timing and number of receptors on the cellular surface—partly controlled by vesicular trafficking through the Golgi and degradation through the endo/lysosomal pathway—is pivotal. Cell-extrinsic signals, such as secreted ligands from the immediate microenvironment, are another critical part of the regulatory landscape of HSC generation. Our work indicates that the availability of those ligands to bind to their cognate receptors at the cell surface is further modulated by the extracellular matrix and its complexity. Understanding the regulatory network that allows for de novo generation of HSCs during embryonic development is crucial to improve our current efforts to produce these precious cells in vitro, with wider significance for stem cell therapies and regenerative medicine.