Plectin, a widespread intermediate filament-based cytolinker protein capable of interacting with a variety of cytoskeletal structures and plasma membrane-bound junctional complexes, serves essential functions in maintenance of cell and tissue cytoarchitecture. We have generated a mouse line bearing floxed plectin alleles and conditionally deleted plectin in stratified epithelia. This strategy enabled us to study the consequences of plectin deficiency in this particular type of tissues in the context of the whole organism without plectin loss affecting other tissues. Conditional knockout mice died early after birth, showing signs of starvation and growth retardation. Blistering was observed on their extremities and on the oral epithelium after initial nursing, impairing food uptake. Knockout epidermis was very fragile and showed focal epidermal barrier defects caused by the presence of small skin lesions. Stratification, proliferation and differentiation of knockout skin seemed unaffected by epidermis-restricted plectin deficiency. In an additionally generated mouse model, tamoxifen-induced Cre-ERT-mediated recombination led to mice with a mosaic plectin deletion pattern in adult epidermis, combined with microblister formation and epidermal barrier defects. Our study explains the early lethality of plectin-deficient mice and provides a model to ablate plectin in adult animals which could be used for developing gene or pharmacological therapies.