Hematopoietic stem cells (HSCs) are pluripotent cells that give rise to all of the circulating blood cell types. Their unique ability to self-renew while generating differentiated daughter cells permits HSCs to sustain blood cell production throughout life. In mammals, the pool of HSCs shifts from early sites in the aorta-gonad-mesonephros region and the placenta to the fetal liver and ultimately bone marrow. During the past decade, a map of transcriptional activators and repressors that regulate gene expression in HSCs, their precursors and their progeny, at distinct stages of development has been drafted. These factors control a program that first establishes the pool of HSCs in the fetus, and later guides decisions between quiescence, self-renewal, and lineage commitment with progressive differentiation to maintain homeostasis. Continuing studies of the regulatory mechanisms that control HSC gene expression followed by the identification of specific loci that are activated or silenced during the life of an HSC will help to further elucidate longstanding issues in HSC decisions to self-renew or to differentiate, and to define the origins of and connections between distinct HSC pools and their precursors.