Human epidermal stem cells express higher levels of β1 integrins and are more adhesive than keratinocytes that are destined to differentiate. To investigate whether high β1 integrin expression and adhesiveness are essential for maintaining keratinocytes in the stem cell compartment, we introduced a dominant-negative β1 integrin mutant, CD8β1, into cultured human keratinocytes, thereby interfering with β1 integrin function. Surface β1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on fibronectin were reduced, and exit from the stem cell compartment was stimulated. Adhesiveness and proliferative potential were restored by overexpressing wild-type β1 integrin or by constitutive MAP kinase activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant decreased adhesiveness and stem cell number in the absence of CD8β1. MAP kinase activation by α6β4-mediated adhesion and mitogens was normal in CD8β1 cells, and constitutive MAP kinase activation did not affect adhesion and proliferation of control keratinocytes. We conclude that β1 integrins and MAP kinase cooperate to maintain the epidermal stem cell compartment in vitro.