Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte-macrophage progenitors (GMPs), whereas differentiation into megakaryocyte-erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1(+)Mac-1(+) myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca(2+) chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte-monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.