Developing standardized bioengineered constructs that accurately replicate human skin is a largely sought-after goal. Pathways initiated at the nurturing interface with the dermal compartment have the potential to modulate the developing epidermal architecture. Here, we identified ascorbic acid, a dermis-donated metabolite, as key in modulating the phenotypical identity of immortalized keratinocytes. Priming monolayers with 2 μg/mL of the culture-stable derivative L-ascorbic acid 2-phosphate (A2P) led to the emergence of a basal-like phenotype within the cells, which showed increased clonogenicity, nuclear/cytoplasmic ratio, and upregulation of progenitor markers. Instead, surpassing this dose induced intracellular ascorbic acid accumulation and promoted a motile status. In organotypic cultures, pre-incubation of founding keratinocytes with 2 μg/mL of A2P improved epithelial layering, whereas higher pretreatments resulted in poor stratification. These findings suggest that ascorbic acid levels in the self-renewing epithelium have a fundamental role in determining whether cells initially commit to differentiation, ultimately influencing regenerative outcomes.