There is increasing evidence to suggest that skin mast cell function is crucially regulated by neuroendocrine controls such as corticotropin-releasing hormone (Ito et al, J Invest Dermatol, 2009) as well as both exo- and endocannnabinoids (Telek et al, FASEB J 2007). We have previously shown that locally produced prototypic endocannabinoids (e.g. anandamide, AEA) markedly inhibit the growth of organ-cultured human hair follicles (HFs) via cannabinoid receptor 1 (CB1). Since perifollicular MCs are important regulators of murine hair growth, we have now investigated the role of CB1 signaling in normal connective tissue MCs (CTS-MCs) in the HF, utilizing human HF organ culture, which is rich in c-Kit+ and CB1+ MCs as a model. While AEA and the CB1-specific agonist ACEA did not change the number or degranulation of CTS-MCs, the CB1-specific antagonist AM251 significantly increased the number of histochemically and immunohistologically detectable CTS-MCs and stimulated their degranulation. Furthermore, AM251 was shown to be directly taken up by c-Kit+ cells, and also upregulated stem cell factor mRNA expression in the HF. siRNA-mediated knockdown of CB1 in HF organ-culture significantly increased the number of both degranulated and total CTS-MCs. Finally, CB1 agonists counteracted the activating effects of MC secretagogues (substance P and compound 48/80) on CTS-MC in situ. These data suggest that endocannabinoid- and CB1-dependent signaling is crucial not only for avoiding excessive MC stimulation but also excessive skin MC maturation from resident precursors. Furthermore, we also show here the first evidence that gene knockdown is possible in an intact, human organ ex vivo. Therefore, from our results, human HFs offers an excellent physiologically and clinically relevant model system for investigating the biology of human CTS-MCs within their natural tissue context.