In vertebrates, hematopoietic stem cell (HSC) emergence mainly occurs in the dorsal aorta of the embryo. It is now well accepted that HSCs arise from hemogenic endothelial (HE) cells via an endothelial-to-hematopoietic transition (EHT) and the formation of intra-aortic hematopoietic clusters (IAHCs). The molecular events controlling endothelial specification, EHT and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells and whole IAHCs isolated from mouse embryo aortas was performed at embryonic day (E)10 and E11. Our analysis identified the genes and transcription factor networks activated during the EHT switch and IAHC cell maturation towards a HSC fate. We also provide an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood related disorders.