Neural stem cells can generate in vitroprogenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca²⁺ signaling controls differentiation of neural precursors. We found low-frequency global and local Ca²⁺ transients occurring predominantly during early stages of differentiation. Spontaneous Ca²⁺signals in individual precursors were not synchronized with Ca²⁺ transients in surrounding cells. Experimentally induced changes in the frequency of local Ca²⁺signals and global Ca²⁺ rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca²⁺ signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype.