Homozygous loss of function of Runx1 during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we employed a conditional gene targeting strategy to effect loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, although there were a number of significant hematopoietic abnormalities observed. Runx1 excision had significant lineage specific effects on B- and T-cell maturation, as well as pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1 deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis comprised of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and provides insights into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies.