Mice lacking the α6 integrin chain die at birth with severe skin blistering. To further study the function of α6 integrin in skin, we generated conditionally immortalized cell lines from the epidermis of wild-type and α6 deficient mouse embryos. Mutant cells presented a decreased adhesion on laminin 5, the major component of the basement membrane in the skin, and on laminins 10/11 and 2. A DNA array analysis revealed alterations in the expression of extracellular matrix (ECM) components including laminin 5, cytoskeletal elements, but also membrane receptors like the hemidesmosomal components integrin β4 and collagen XVII, or growth factors and signaling molecules of the TGFβ, EGF, and Wnt pathways. Finally, an increase of several epidermal differentiation markers was observed in cells and tissue at the protein level. Further examination of the mutant tissue revealed alterations in the filaggrin signal. These differences may be linked to an upregulation of the TGFβ and the Jun/Fos pathways in mutant keratinocytes. These results are in favor of a role for integrin α6β4 in the maintenance of basal keratinocyte properties and epidermal homeostasis.