Hair follicle neural crest stem cells reside in the bulge region of the outer root sheath of hair follicles, originate from the ectoderm, and have multidirectional differentiation potential, making them ideal candidates for tissue engineering applications. These cells mainly reside in a hypoxic microenvironment that favors the maintenance of stemness. Recently, many studies have elucidated the involvement of the Hippo pathway in the regulation of stem cell fate. However, few studies have investigated whether the Hippo signaling pathway regulates the growth of hair follicle neural crest stem cells in hypoxic environments. In the present study, we investigated the role of the Hippo pathway in the regulation of hair follicle neural crest stem cells under hypoxic conditions. We identified neural crest-derived stem cells from single-cell RNA-seq data of skin organoids in a public database, and reported that the Hippo pathway was activated in the cell population. Hair follicle neural crest stem cells were isolated from rat hair follicles and cultured under hypoxic (3% oxygen) and normoxic (20% oxygen) conditions. Cell viability was assessed via the CCK8 assay. The expression levels of several key genes, including Hif2α, Nestin, Sox10, Oct4, Nanog, Sox2, and Klf4, were evaluated via quantitative real-time PCR, after which we treated the cells with verteporfin, a small molecule inhibitor of the Hippo pathway. Changes in the subcellular localization of the hair follicle neural crest stem cell-specific marker SOX10 were assessed via immunofluorescence. Western blotting was used to analyze the expression levels of proteins associated with stemness and hypoxia responses, including HIF2α, SOX10, OCT4, NANOG, SOX2, and KLF4. The results showed that hypoxic conditions facilitated the maintenance of stemness in hair follicle neural crest stem cells, including the promotion of proliferation and the expression of multipotential markers. Inhibition of the Hippo pathway results in a significant decrease in cell proliferation. The protein expression of HIF2α, SOX10, OCT4, NANOG, SOX2, and KLF4 was also reduced under hypoxic conditions.