Directing neural stem cells (NSCs) differentiation towards oligodendroglial cell lineage is a crucial step in the endeavor of developing cell replacement-based therapies for demyelinating diseases. Evaluation of NSCs differentiation is mostly performed by methodologies that use fixed cells, like immunocytochemistry, or lysates, like Western blot. On the other hand, electrophysiology allows differentiation studies on living cells, but it is highly time-consuming and endowed with important limitations concerning population studies. Herein, we describe a functional method, based on single cell calcium imaging, which accurately and rapidly distinguishes cell types among NSCs progeny, in living cultures prepared from the major reservoir of NSCs in the postnatal mouse brain, the subventricular zone (SVZ). Indeed, by applying a rational sequence of three stimuli-KCl, histamine, and thrombin-to the heterogeneous SVZ cell population, one can identify each cell phenotype according to its unique calcium signature. Mature oligodendrocytes, the myelin-forming cells of the central nervous system, are the thrombin-responsive cells in SVZ cell culture and display no intracellular calcium increase upon KCl or histamine perfusion. On the other hand, KCl and histamine stimulate neurons and immature cells, respectively. The method described in this chapter is a valuable tool to identify novel pro-oligodendrogenic compounds, which may play an important role in the design of future treatments for demyelinating disorders such as multiple sclerosis.