Summary Mammalian neural stem/progenitor cells (NSPCs) sequentially generate neurons and glia during CNS development. Here we identified miRNA-153 (miR-153) as a modulator of the temporal regulation of NSPC differentiation. Overexpression (OE) of miR-153 delayed the onset of astrogliogenesis and maintained NSPCs in an undifferentiated state in vitro and in the developing cortex. The transcription factors nuclear factor I (NFI) A and B, essential regulators of the initiation of gliogenesis, were found to be targets of miR-153. Inhibition of miR-153 in early neurogenic NSPCs induced precocious gliogenesis, whereas NFIA/B overexpression rescued the anti-gliogenic phenotypes induced by miR-153 OE. Our results indicate that miR-mediated fine control of NFIA/B expression is important in the molecular networks that regulate the acquisition of gliogenic competence by NSPCs in the developing CNS.Gliogenesis is tightly inhibited during the early neurogenic phase of central nervous system development. Okano, Shimazaki, and colleagues identified a microRNA, miR-153, as an inhibitor of gliogenesis in early neurogenic neural stem/progenitor cells (NSPCs). This study demonstrates that miR-153 controls the timing of the acquisition of gliogenic competence via direct repression of the gliogenic transcription factors NFIA and NFIB.