Even though the involvement of intracellular Ca2+ () in hematopoiesis has been previously demonstrated, the relationship between signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca2+ signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP3) levels and release in murine and human hematopoietic stem/progenitor cells. In addition, signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca2+-dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca2+ release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca2+ signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells. J. Cell. Physiol. 226: 1780–1792, 2011. © 2010 Wiley-Liss, Inc.