Hematopoietic stem cells sequentially pass through a series of decision points affecting self-renewal or lineage-specific differentiation. Notch1 receptor is a known modulator of lineage-specific events in hematopoiesis that we assessed in the context of in vivo stem cell kinetics. Using RAG-1(-/-) mouse stems cells, we documented increased stem cell numbers due to decreased differentiation and enhanced stem cell self-renewal induced by Notch1. Unexpectedly, preferential lymphoid over myeloid lineage commitment was noted when differentiation occurred. Therefore, Notch1 affects 2 decision points in stem cell regulation, favoring self-renewal over differentiation and lymphoid over myeloid lineage outcome. Notch1 offers an attractive target for stem cell manipulation strategies, particularly in the context of immunodeficiency and acquired immunodeficiency syndrome.