The α6β4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of α6β4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that α6β4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the β4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of β4 on various biological processes by comparing β4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking α6β4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of α6β4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes – derived from β4-subunit conditional knockout mice – from which the gene encoding β4 had been deleted by Cre-mediated recombination. Consistent with the established role of α6β4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of α6β4, provided that cell adhesion is not compromised.