Activity-induced neurogenesis via Ca(2+) entry may be important for establishing Hebbian neural network. However, it remains unclear whether intracellular Ca(2+) mobilization is required and which subtypes of Ca(2+) release channels expressed in Ca(2+) store organelles are involved in the activity-dependent neurogenesis. Here, we demonstrated that the activity of intracellular Ca(2+) signaling, expression of neuronal transcription factor NeuroD, and the rate of neurogenesis were significantly inhibited in neuronal cells derived from embryonic stem (ES) cells deficient in the Ca(2+) release channel type 2 ryanodine receptors (RyR2(-/-)). In wild-type (RyR2(+/+)) but not in RyR2(-/-) ES cells, activation of L-type Ca(2+) channels, GABA(A) receptors, or RyRs promoted neuronal differentiation, while inhibition of these channels/receptors had an opposite effect. Moreover, neuronal differentiation promoted by activation of GABA(A) receptors or L-type Ca(2+) channels in RyR2(+/+) cells was prevented by RyR inhibitors. No significant difference was detected in the expression level of GABA(A) receptors and L-type channels between neuronal cells derived from two types of ES cells. Thus, activity-induced Ca(2+) influx through L-type Ca(2+) channels alone is not sufficient in promoting neurogenesis. Instead, an intimate cooperation of L-type Ca(2+) channels with RyR2 is crucial for the activity-dependent neurogenesis induced by paracrine and/or autocrine GABA signaling.