At the early postnatal period, cerebellar granule cells proliferate, differentiate, migrate, and finally form refined synaptic connections with mossy fibers. During this period, the resting membrane potential of immature granule cells is relatively depolarized, but it becomes hyperpolarized in mature cells. This investigation was conducted to examine the role of this alteration in membrane potential and its downstream signaling mechanism in development and maturation of granule cells. Experiments were designed to precisely characterize the ontogenic processes of developing granule cells by combining organotypic cerebellar cultures with the specific expression of EGFP (enhanced green fluorescent protein) in granule cells by use of DNA transfection. Multiple approaches using morphology, electrophysiology, and immunohistochemistry demonstrated that granule cells developed and matured at the physiological KCl concentration in organotypic cultures in a temporally regulated manner. We addressed how persistent membrane depolarization influences the developmental and maturation processes of granule cells by depolarizing organotypic cultures with high KCl. Depolarization preserved the developmental processes of granule cells up to the stage of formation of immature dendrites but prevented the maturation processes for synaptic formation by granule cells. Importantly, this blockade of the terminal maturation of granule cells was reversed by inactivation of calcineurin with its specific inhibitor. This investigation has demonstrated that alteration of the membrane potential and its downstream calcineurin signaling play a pivotal role in triggering the maturation program for the synaptic organization of postnatally developing granule cells.