Reprogramming of gene expression by post-translational modification of chromatin is a hallmark of cellular differentiation. In particular, histone acetylation states in the cell, which are determined by a balance in the activities of histone acetyltransferases (HATs) and histone deacetylases, reflect the highly dynamic control of transcriptional activation and repression. In this study, I characterized the functions of two highly-related HAT paralogues, lysine acetyltransferase 2A (KAT2A) and lysine acetyltransferase 2B (KAT2B), in regulating the proliferation and differentiation of human keratinocytes. I found that keratinocytes switch from high KAT2A and low KAT2B expression under self-renewing conditions, to low KAT2A and high KAT2B expression upon cellular differentiation. In proliferative keratinocytes, depletion of KAT2A triggered changes in cell morphology, colony clustering, perturbation of cell trajectories, and led to premature expression of early and late differentiation markers in the absence of initiating signals. By contrast, KAT2B loss did not have a significant impact on the biology of proliferative keratinocytes. The effects of KAT2A depletion in self-renewing keratinocytes coincided with a specific and extensive loss of global H3K9ac levels, a histone modification known to affect transcriptional elongation. In addition, I found that the function of KAT2A in proliferating keratinocytes is wholly dependent on its acetyltransferase activity as I was able to rescue the aberrant culture morphology and premature differentiation of KAT2A-depleted cells with the expression of full-length KAT2A but not with an acetyltransferase-dead mutant isoform. On induction of differentiation, KAT2A-deficient cells differentiated rapidly in response to initial signals whilst KAT2B-depleted keratinocytes exhibited delayed differentiation morphology and decreased expression of mid-to late differentiation markers. Taken together, my results indicate that KAT2A functions primarily to support the maintenance of keratinocyte stemness, whilst KAT2B acts to promote cellular differentiation. These findings revealed a distinctive gene regulatory mechanism in which keratinocytes utilise a pair of highly homologous HATs to support divergent functions in stem cell self-renewal and differentiation.