HaCaT cell line is a widely used model for studying normal human keratinocytes. However, mutations of TP53 gene are typical for this cell line, which have a substantial impact on functions of the encoded protein. The features of this regulatory circuit should be considered when using HaСaT cells for assessment of human skin physiology and pathology in vitro. The study was aimed to assess the features of differentiation realization in HaCaT cells with modulated activity of p53 protein. The expression of р53 was reduced by knockdown of ТР53 gene by shRNA (by 2.2 times, p < 0.05), and the elevated concentration of the р53 active forms was achieved via exposure of cells to Nutlin-3a, the MDM2 inhibitor and the major negative regulator of р53. It has been found that regulation of at least three differentiation markers, СASP14, IVL (expression increase by 3.9 and 3.7 times respectively in the p53-knockdown cells, p < 0.05) and TGM1 (twofold expression decrease in the p53-knockdown cells, and 1.7-fold expression increase under exposure to Nutlin-3a, p < 0.05) in HaCaT cells is p53-mediated. The positive correlation has been revealed for expression of TGM1 and p53 that might be realized indirectly via ΔNp63 expression alteration. At the same time, modulation of p53 does not result in significant alterations in expression of cytokeratins.