Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent NADP analogue that acts as an intracellular calcium mobilizing messenger by targeting lysosomal-like calcium stores. In this study, we compared the effects of NAADP with those of the prototypical messenger inositol trisphosphate on cytosolic calcium levels and differentiation of PC12 cells and neural stem cells. We demonstrate that liposomal delivery of NAADP mediated a robust and sustained increase in cytosolic calcium in PC12 cells and neural stem cells (isolated from olfactory bulb and subventricular zone of postnatal rat brain). The NAADP-induced calcium signal had the pharmacological characteristics of the calcium release from acidic calcium stores. This stimulus was sufficient to drive differentiation of the cells to a neuronal-like phenotype. However, in contrast, the cell fate was unaffected by more transient calcium signals generated by inositol trisphosphate-evoked release of endoplasmic reticulum calcium stores. Our data establish for the first time (i) the presence of novel NAADP-sensitive calcium stores in PC12 cells and neural stem cells, (ii) a role for NAADP in differentiation, and (iii) that calcium-dependent function can be messenger-specific. Thus, differential recruitment of intracellular calcium mobilizing messengers and their target calcium stores may represent a robust means of maintaining stimulus fidelity in the control of calcium-dependent cell function.