In this study, we examined the role of PKC in the differentiation of multipotential neural precursor cells (NPCs). We found that the NPCs expressed PKCalpha,beta2,delta,epsilon,zeta and low levels of PKCgamma. The PKC activator, PMA, selectively increased the number of astrocytes, whereas it decreased the generation of neurons and oligodendrocytes. Similarly, overexpression of PKCepsilon increased the differentiation of astrocytes and a PKCepsilonKD mutant abolished PMA effect. PMA phosphorylates PKCepsilon on serine 729. Using a PKCepsilonS729A mutant, we found that the phosphorylation of PKCepsilon on serine 729 was essential for the differentiation of astrocytes induced by PMA. To delineate the mechanisms involved in PMA and PKCepsilon effects, we examined the expression of Notch1, which has been associated with astrocytic differentiation. We found that PMA and PKCepsilon induced a large increase in Notch1 expression and the PKCepsilonS729A mutant abolished PMA effect. Moreover, the PKCepsilonS729A mutant also inhibited the effect of CNTF on astrocytic differentiation and Notch 1 expression. Finally, Notch1 mediated the effect of PMA on astrocytic differentiation, since the gamma-secretase inhibitor L-685,458, and Notch1 silencing abolished PMA effect. Our data suggest an important role of PKCepsilon in astrocytic differentiation and implicate Notch1 as a possible mediator of this effect.