It is known that cerebellar granule cells are powerful inducers for the differentiation of Purkinje cells. However, the detailed mechanism of this regulation has not yet been clarified. Here, using cerebellar neuronal culture, we show that the activation of NMDA receptors expressed by granule cells triggers the signaling pathway for the dendritic differentiation of Purkinje cells. This signal has been shown to promote the granule cell survival through BDNF-mediated TrkB activation, leading to Purkinje cell differentiation by increasing the granule-Purkinje cell interaction. Among the possible signal molecules provided to the dendrites of Purkinje cells from granule cells, nitric oxide was found to have no effect on the dendritic outgrowth and branching, but electrical activity and the subsequent intracellular Ca(2+) increase were thought to play an important role in the branching and thickening of the dendrites, because blockade of both non-NMDA and metabotropic glutamate receptors caused a significant decrease in the number of branch points and the diameter of Purkinje dendrites without apparently affecting the dendrite extension and spine formation. Collectively, these results suggest that Purkinje cell differentiation is regulated by two successive steps. The first step is initiated by the NMDA receptor-mediated signal in granule cells, which acts as a trophic factor for granule cells. The second step involves the activation of granule-Purkinje synapses, providing neurotrophic substances and electrical activity essential for Purkinje cell differentiation.