During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally lose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal-specific protein that binds Src kinase and protein kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src kinase-dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons.