Granulocyte colony-stimulating factor (G-CSF) plays a major role in the regulation of granulopoiesis. Treatment of cells with G-CSF has been shown to activate multiple signal transduction pathways. We show here that Erk5, a novel member of the MAPK family, and its specific upstream activator MEK5 were activated in response to incubation of cells with G-CSF. Different from other members of the MAPK family including Erk1/2, JNK, and p38, maximal activation of Erk5 by G-CSF required the C-terminal region of the G-CSF receptor. Genistein, a specific inhibitor of protein-tyrosine kinases, blocked G-CSF-induced Erk5 activation. In contrast, inhibition of protein kinase C activity increased G-CSF-mediated activation of Erk5 and MEK5, whereas stimulation of protein kinase C activity inhibited activation of the two kinases by G-CSF. The proliferation of BAF3 cells in response to G-CSF was inhibited by expression of a dominant-negative MEK5 but potentiated by expression of a constitutively active MEK5. Expression of the constitutively active MEK5 also increased the survival of BAF3 cells cultured in the absence of or in low concentrations of G-CSF. Together, these data implicate Erk5 as an important signaling component in the biological actions of G-CSF.