Numb is an evolutionarily conserved protein that controls the differentiation of neuronal progenitor cells by unknown mechanisms. Here we report that the neural cells expressing Numb isoforms with short phosphotyrosine-binding (SPTB) domain undergo extensive neurite outgrowth, an effect that can be blocked by voltage-gated Ca2+ channel (VGCC) inhibitor or by Ca2+ chelator. In contrast, tyrosine kinase inhibitor, genistein, and selective receptor tyrosine kinase (TrkA) inhibitor, K252alpha did not affect SPTB Numb-mediated neurite outgrowth. MAP kinase inhibitor, PD98059 partially reduced SPTB Numb-mediated neurite outgrowth. Cells expressing SPTB Numbs exhibit increased whole-cell Ca2+ current densities (ICa) which can be prevented by preincubation of either nifedipine or PD98095. Cells expressing LPTB Numbs expressed little ICa (density) and were not able to grow neurites. Our results indicate that Ca2+ influx through VGCC may be required for SPTB Numb-mediated neurite outgrowth, suggesting that Numb promotes neuronal differentiation by a mechanism involving PTB domain-specific regulation of Ca2+ influx and MAP kinase activation.