In order to develop an efficient strategy to induce the in vitro differentiation of neural stem cells (NSCs) into oligodendrocyte progenitor cells (OPCs), NSCs were isolated from E14 mice and grown in medium containing epidermal growth factor and fibroblast growth factor (FGF). Besides supplementing the medium with oligodendrogenic factors such as Sonic Hedgehog (Shh), FGF-2, and PDGF, we attempted to initiate the gene transcription program for OPC differentiation by transfection of the Olig1 gene, a transcription factor known to be involved in the induction of oligodendrocyte lineage formation during embryogenesis. Whereas addition of Shh, FGF-2, and PDGF could induce OPC differentiation in 12% of the NSCs, the transient expression of Olig1 by use of Nucleofector gene transfection initiated OPC differentiation in 55% of the NSCs. Our results show that nonviral transfection of genes encoding for oligodendrogenic transcription factors may be an efficient way to initiate the in vitro differentiation of NSCs into OPCs.