Abstract Keratins are intermediate filament (IF) forming proteins whose expression and post-translational modifications are tightly regulated in response to development, differentiation, stress, and disease. Keratin 14 (K14) is highly expressed in progenitor keratinocytes located in the basal layer of stratified squamous epithelia, including the skin epidermis. K14 and its obligate assembly partner keratin 5 (K5) form highly stable coiled-coil dimers that further polymerize into 10 nm keratin IF filaments in basal keratinocytes. The coiled-coiled structure of all IF proteins including K5/K14 heterodimers is made possible by long-range heptad repeats located within their defining, central rod domain. All IF proteins contain a 4-residue disruption in this heptad repeat at a specific location within the rod domain, known as the stutter region. K14 and select keratins expressed in surface epithelia contain an evolutionarily conserved cysteine residue in position 2 within the stuffer motif (mouse K14 C373). The functions of the stutter region and its conserved cysteine remain incompletely defined. Recent work in the Coulombe lab identified a role for K14 stutter cysteine in the regulation of epidermal homeostasis. Expanding upon prior a crystal structure determination and follow-up observations, we generated a Krt14 C373A mouse line, which shows altered K14-dependent disulfide bonding in the epidermis. The remarkable molecular phenotype of the Krt14 C373A mouse informed our current mechanism proposing that disulfide bonded K14 binds to the scaffolding protein 14-3-3σ, in a differentiation cue-dependent fashion, leading to sequestration and inactivation of the pro-growth transcriptional co-activator YAP1. As part of ongoing studies, we sought to accentuate the otherwise subtle Krt14 C373A gross phenotype, in contrast to its molecular phenotype, by generating a novel mouse line, Krt14 C373A/null. The Krt14 C373A/null mouse contains a single mutant Krt14 allele, resulting in reduced levels of K14 post-translationally modified at the stutter. The Krt14 C373A/null mouse phenotype presents with precocious post-natal development, hair follicle abnormalities, and crowded, irregular basal keratinocyte nuclei in the skin and tongue. From observations made so far using Krt14 C373A/null mice I hypothesize that the concentration and proportion of stutter cysteine-dependent disulfide bonded K14 within the keratin IF network of basal keratinocytes define basal keratinocyte identity and regulate their entry into terminal differentiation. Further, I hypothesize that alterations in the regulation of basal cell identity will promote skin cancer initiation and progression. This hypothesis proposes that basal keratinocyte identity is defined by the composition of the keratin filament network, including K14 and an additional type I keratin, K15, and that the proper balance of K15 and disulfide-bonded K14 prevents pathological growth of progenitor keratinocytes. Altogether ...