Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at “stemness” genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating “stemness” genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.