Located at the interface between body and environment, the epidermis must protect the body against toxic agents and dehydration, and protect itself against physical and mechanical stresses. Acquired just before birth and at the last stage of epidermal differentiation, the skin's proteinaceous/lipid barrier creates a surface seal essential for protecting animals against microbial infections and dehydration. We show here that Kruppel-like factor 4 (Klf4, encoded by the gene Klf4), highly expressed in the differentiating layers of epidermis, is both vital to and selective for barrier acquisition. Klf4-/- mice die shortly after birth due to loss of skin barrier function, as measured by penetration of external dyes and rapid loss of body fluids. The defect was not corrected by grafting of Klf4-/- skin onto nude mice. Loss of the barrier occurs without morphological and biochemical alterations to the well-known structural features of epidermis that are essential for mechanical integrity. Instead, late-stage differentiation structures are selectively perturbed, including the cornified envelope, a likely scaffold for lipid organization. Using suppressive subtractive hybridization, we identified three transcripts encoding cornified envelope proteins with altered expression in the absence of Klf4. Sprr2a is one, and is the only epidermal gene whose promoter is known to possess a functional Klf4 binding site. Our studies provide new insights into transcriptional governance of barrier function, and pave the way for unravelling the molecular events that orchestrate this essential process.