Several leukocyte populations normally reside in mouse skin, including Langerhans cells and γδ T cells in the epidermis and macrophage and mast cells in the dermis. Interestingly, these skin resident leukocytes are frequently identified within or around hair follicles (HFs), which are known to contain stem cell populations that can generate the epidermal architecture or give rise to the melanocyte lineage. Thus, we reasoned that HFs might serve as a local reservoir of the resident leukocyte populations in the skin. When vibrissal follicles of adult mice were cultured in the presence of stem cell factor (SCF), interleukin 3 (IL-3), IL-7, granulocyte-macrophage colony-stimulating factor, and Flt3 ligand, CD45⁺/lineage^–/c-kit⁺/FcϵRI⁺ cells became detectable on the outgrowing fibroblasts in 10 days and expanded progressively thereafter. These HF-derived leukocytes showed characteristic features of connective tissue-type mast cells, including proliferative responsiveness to SCF, metachromatic granules, mRNA expression for mast cell proteases-1, -4, -5, and -6, and histamine release on ligation of surface IgE or stimulation with substance P or compound 48/80. These results, together with our findings that HFs contain c-kit⁺ cells and produce SCF mRNA and protein, suggest that HFs provide a unique microenvironment for local development of mast cells.