Stemness of epidermal stem cells(ESCs) is the result of the combination of intrinsic characteristics and extrinsic microenvironment. The key point of in vitro culturing ESC in long term is maintaining the elements of microenvironment. This experiment isolates ESC from the cells originating from skin explant cultures through respective trypsinization and type IV collagen rapid adhering. And ESCs are cultured in serum-free medium containing 20% conditioned medium. The original cells show K19 positive immunofluorescence staining, and form PGCs colony-like cell groups. Cells originating from both epidermis and dermal tissue show typical characteristics, colony forming and road metal-like shape. They are respectively passaged to 5 and 8 passage. Passaged cells showβ1 integrin positive immunofluorescence staining. The colony-forming efficiency (CFE) of passage 5 and passage 8 are 18.5% and 8.5%. All these results demonstrate that skin explant culturing method can maintain the microenvironment of ESC, and the harvested ESC show characteristics of stemness and can be stably passaged.