Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are up-regulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (TNF-α) cytokines on epidermal morphogenesis, proliferation, differentiation and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31 and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.Journal of Investigative Dermatology accepted article preview online, 11 February 2014; doi:10.1038/jid.2014.83.