Chemical biology focuses on the discovery and use of chemical compounds to understand and manipulate biological systems. It has been applied in many fields, including stem cell research. Recently, stem cell research has made significant progresses. A better understanding of stem cell biology will also promote the translation of stem cell technology in regenerative medicine. Elucidating the mechanisms governing cell fate determination and developing strategies to precisely manipulate cell fates are the fundamental issues in stem cell research. The use of small molecules to manipulate stem cell behaviors, including self-renewal, differentiation and reprogramming, has become more and more popular and offered significant advantages over conventional genetic approaches for their convenience in use, high degree of temporal control and functional diversity. In addition, based on our long-term experience in small molecular drug development, the application of chemical compounds to modulate stem cell functions in vivo for regeneration and repair could be the future direction of regenerative medicine.