Skin homeostasis is maintained by controlling the balance between proliferation and differentiation of epidermal stem cells. The microenvironment, including extrinsic stresses, growth factors, soluble molecules, cell-ECM and cell-cell communications, plays an important role in cell fate determination in vivo and in vitro. In response to external signals, keratinocytes cooperate with other cell types to modulate and facilitate the wound microenvironment during wound healing; however, the aberrant signals or conjunctions in the environment will lead to pathologic abnormalities. In addition, despite some drawbacks, the epidermal stem-cellbased bioengineered skin substitutes have greatly improved the quality of cutaneous repair. Thus, exploring the characteristics and regulation mechanisms of microenvironment-dependent homeostasis and differentiation of epidermal basal undifferentiated keratinocytes is necessary to understand skin development and wound repair and to design novel therapeutic strategies for skin wound healing.