The role of intracellular Ca2+ (Ca2+i) on hematopoiesis was investigated in long term bone marrow cultures using cytokines and agonists of P2 receptors. Cytokines interleukin 3 and granulocyte/ macrophage colony stimulator factor promoted a modest increase in Ca 2+i concentration ([Ca2+]i) with activation of phospholipase Cγ, MEK1/2, and Ca2+/calmodulin kinase II. Involvement of protein kinase C was restricted to stimulation with interleukin 3. In addition, these cytokines promoted proliferation (20 times) and an increase in the Gr-1-Mac-1+ population with participation of gap junctions (GJ). Nevertheless ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1-Mac-1-c-Kit + population, and differentiation into macrophages without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca 2+]i would be related to cytokine-dependent proliferation with participation of GJ, whereas high increases in [Ca2+] i would be related to macrophage differentiation without maintenance of the primitive population. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.