Hematopoietic Stem Cells (HSCs) are used clinically to treat human blood-related genetic diseases and leukemias, but the availability of this therapy is constrained by the limiting number of transplantable HSCs. Previous strategies to expand HSCs in vitro resulted in precocious differentiation and reduced transplant efficiency. In vivo analysis of the mechanisms utilized by fetal and adult niches to control HSCs can be mimicked for therapeutic use. This review summarizes the latest research on the in vivo HSC niche and the clinical applications of this knowledge to promote ex vivo expansion and direct de novo generation of HSCs.