Erythropoietin (EPO) and stem cell factor (SCF) are key cytokines that regulate the early stages of erythroid differentiation. However, it remains undetermined whether additional cytokines also play role in the differentiation program. Here we report that osteopontin (OPN) is highly expressed and secreted by erythroblasts during differentiation. We also demonstrate that OPN deficient human and mouse erythroblasts exhibit defects in F-actin filaments and addition of exogenous OPN to OPN deficient erythroblasts restored the F-actin filaments in these cells. Furthermore, our studies demonstrate that OPN contributes to erythroblast proliferation. OPN knock-out male mice exhibit lower hematocrit (Hct) and hemoglobin (Hgb)levels compared to their wild type counterparts. We also show that OPN mediates phosphorylation or activation of multiple proteins including Rac-1 GTPase and the actin binding protein, adducin in human erythroblasts. In addition, we show that OPN's effects include regulation of intracellular calcium in human erythroblasts. Finally, we demonstrate that human erythroblasts express CD44 and integrins beta1 and alpha4, three known receptors for OPN and that the integrin beta1 receptor is involved in transmitting the proliferative signal. Together these results provide evidence for signal transduction by OPN and contribution to multiple functions during the erythroid differentiation program in human and mouse.