Stem cell therapy holds great promise for regenerative medicine and the treatment of numerous diseases. A key issue of stem cell therapy is the control of stem cell fate, but safe and practical methods are limited. Essential fatty acids, namely ω‐6 (n‐6) and ω‐3 (n‐3) polyunsaturated fatty acids (PUFA), and their metabolites are critical components of cell structure and function, and could therefore influence stem cell fate. The available evidence demonstrates that n‐6 and n‐3 PUFA and their metabolites can act through multiple mechanisms to promote the proliferation and differentiation of various stem cell types. Therefore, elucidating the role of PUFA and their metabolites in stem cell fate regulation is both a challenge and an opportunity for stem cell biology as well as stem cell therapy. PUFA‐based interventions to create a favorable environment for stem cell proliferation or differentiation may thus be a promising and practical approach to controlling stem cell fate for clinical applications. Stem Cells 2014;32:1092–1098