Hair follicle stem cells (HFSCs), a kind of adult stem cells located in the bulge region of hair follicles, participate in cyclical hair follicle renewal and skin wound repair. Current evidence indicates that Wnt/β-catenin, bone morphogenetic protein (BMP), and sonic hedgehog (Shh) signaling pathways, together with neural, metabolic, mechanical, and injury-related microenvironments, regulate the transition between quiescence and activation in HFSCs. Three-dimensional culture, organoids, gene editing, high-content imaging, and single-cell analysis have also played a promoting role in its functional studies and translational evaluation. HFSCs show potential for hair regeneration, wound repair, and tissue engineering; however, maintenance of stemness, cellular heterogeneity, delivery and integration efficiency, and long-term safety require further investigation. This review summarizes the morphology and distribution, proliferation and differentiation, phenotypic markers, regulatory signals, and research techniques of HFSCs, as well as evidence for their use in hair regeneration, wound repair, tissue engineering, and neural regeneration, providing a reference for mechanistic studies and translational evaluation of HFSC-related strategies.