Because of its multiple effects in tissue homeostasis and cancer, Notch signaling is gaining increasing attention as a potential therapeutic target. Notch proteins belong to a family of highly conserved cell surface receptors. Ligand binding leads to proteolytic cleavage of Notch receptors by the γ-secretase complex, followed by translocation of the active intracellular Notch domain into the nucleus and transcriptional activation. Multiple genetic and pharmacological methods are available to inhibit or activate the Notch pathway, some of which are entering human clinical trials. In this review, we discuss our current understanding of Notch signaling in the hematopoietic system. Canonical Notch signaling is essential for the generation of definitive embryonic hematopoietic stem cells, but dispensable for their maintenance during adult life. Notch controls several early steps of T cell development, as well as specific cell fate and differentiation decisions in other hematopoietic lineages. In addition, emerging evidence indicates that Notch is a potent, context-specific regulator of T cell immune responses, including in several disease models relevant to patients. This knowledge will constitute a framework to explore Notch modulation as a therapeutic strategy and to understand potential hematopoietic side effects of systemic Notch inhibition.