Understanding how complex tissues are formed, maintained, and regenerated through local growth, differentiation, and remodeling requires knowledge on how single‐cell behaviors are coordinated on the population level. The self‐renewing hair follicle, maintained by a distinct stem cell population, represents an excellent paradigm to address this question. A major obstacle in mechanistic understanding of hair follicle stem cell (HFSC) regulation has been the lack of a culture system that recapitulates HFSC behavior while allowing their precise monitoring and manipulation. Here, we establish an in vitro culture system based on a 3D extracellular matrix environment and defined soluble factors, which for the first time allows expansion and long‐term maintenance of murine multipotent HFSCs in the absence of heterologous cell types. Strikingly, this scheme promotes de novo generation of HFSCs from non‐HFSCs and vice versa in a dynamic self‐organizing process. This bidirectional interconversion of HFSCs and their progeny drives the system into a population equilibrium state. Our study uncovers regulatory dynamics by which phenotypic plasticity of cells drives population‐level homeostasis within a niche, and provides a discovery tool for studies on adult stem cell fate.
