Hematopoietic stem cells (HSCs) are at the origin of adult hematopoiesis, providing an organism with all blood cell types needed throughout life. During embryonic development a first wave of hematopoiesis (independent of HSCs) allows the survival and growth of the embryo until birth. A second wave of hematopoiesis that will last into adulthood depends on the production of HSCs that begins at mid-gestation in large arteries such as the aorta. HSC production occurs through a hemogenic endothelial to hematopoietic transition (EHT) process and the formation of hematopoietic clusters in most vertebrate species. Advances in understanding EHT, cluster formation and HSC production were triggered by combined progresses made in the development of in vivo assays, microscopy, imaging and fluorescence tools. Here, we review the current knowledge on developmental hematopoiesis with a focus on the first step of HSC production in the aorta and how microscopic approaches have contributed to a better understanding of the vital process of blood cell formation.