Wnts are deeply involved in the proliferation and differentiation of skin epithelial cells. We previously reported the differentiation of cultured primary skin epithelial cells toward hair shaft and inner root sheath (IRS) of the hair follicle via β-catenin stabilization caused by Wnt-10b, however, the effects of Wnt-10b on cultured hair follicles have not been reported. In the present study, we examined the effects of Wnt-10b on shaft growth using organ cultures of whisker hair follicles in serum-free conditions. No hair shaft growth was observed in the absence of Wnt-10b, whereas its addition to the culture promoted elongation of the hair shaft, intensive incorporation of BrdU in matrix cells flanking the dermal papilla (DP), and β-catenin stabilization in DP and IRS cells. These results suggest a promoting effect of Wnt-10b on hair shaft growth that is involved with stimulation of the DP via Wnt-10b/β-catenin signalling, proliferation of matrix cells next to the DP, and differentiation of IRS cells by Wnt-10b.