We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27kip1 [H. J. Kim et al. (2006)Brain Research, 1092, 1–15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 μm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-β1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-β1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-β1 (10 ng/mL) did not increase neurite outgrowth but rather the number of β-tubulin III-positive cells, which was accompanied by enhanced p27kip1 mRNA expression. In addition, TGF-β receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-β1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-β1 signaling inhibition, via a TGF-β receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27kip1 protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-β1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27kip1 activation in NPCs.