The grainy head (grh) transcription factor is a critical regulator for the formation and maintenance of the cuticle in Drosophila. The developmental roles of grh have been subfunctionalised across the mammalian genes, with the Grhl3 gene most pivotal for epidermal integrity. Constitutive loss of Grhl3 leads to neonatal lethality due to failure of skin barrier formation. The key Grhl3 target gene in this process is Transglutaminase 1 (Tgm1), for cross-linking the lipid-protein matrix in the stratum corneum. Intriguingly, conditional loss of Grhl3 in the epidermis after barrier function is established does not lead to barrier regression. This finding prompted the search for compensatory factors in barrier maintenance post-natally. Based on expression patterns, we postulated that Grhl1 may be this factor, and we initiated breeding strategies to generate conditional Grhl3-null mice that were constitutively lacking Grhl1. Pups with this genotypedeveloped a normal skin barrier late in gestation as evidenced by dye exclusion and transepidermal water loss analysis. However, none of these mice survived to weaning. Time of death analysis revealed that all animals succumb in the first 48 hours of life due to excessive trans-epidermal water. Phylogenetic analysis coupled with gene expression profiling identified Transglutaminase 5 (Tgm5) as the critical Grhl1 target required with Tgm1 for maintenance of the epidermal barrier in the terrestrial environment. These studies represent the first example of genetically-induced loss of pre-established skin barrier function and identify the Grhl factors as potential therapeutic targets in human diseases in which the skin barrier function is compromised, such as atopic dermatitis, and psoriasis.