The defined location of a stem cell within a niche regulates its fate, behavior and molecular identity via a complex extrinsic regulation far from being fully elucidated yet. To explore the molecular characteristics and key components of the aortic microenvironment, where the first hematopoietic stem cells (HSCs) are generated during development, we performed genome-wide RNA tomography sequencing on zebrafish, chicken, mouse and human embryos. The resulting anterior-posterior and dorsal-ventral transcriptional maps provided a powerful resource for exploring genes and regulatory pathways active in the aortic microenvironment. By performing inter-species comparative RNA-seq analyses and functional assays, we explored the complexity of the aortic microenvironment landscape and the fine-tuning of various factors interplaying to control HSC generation both in time and space in vivo, including the ligand-receptor couple ADM-RAMP2 and SVEP1. Understanding the regulatory function of the local environment will pave the way for improved stem cell production in vitro and clinical cell therapy.