Transforming growth factor β1 (TGF-β1) plays a role in the maintenance of quiescent hematopoietic stem cells (HSCs) in vivo. We asked whether TGF-β1 controls the cell cycle status of HSCs in vitro to enhance the reconstitution activity. To examine the effect of TGF-β1 on the HSC function, we used an in vitro culture system in which single HSCs divide with the retention of their short- and long-term reconstitution ability. Extensive single-cell analyses showed that, regardless of its concentration, TGF-β1 slowed down the cell cycle progression of HSCs but consequently suppressed their self-renewal potential. Cycling HSCs were not able to go back to quiescence with TGF-β1. This study revealed a negative role of TGF-β1 in the regulation of the HSC number and reconstitution activity.