Balance between the open and locked structure of chromatin is orchestrated by posttranslational histone modifications. Acetylation of the histone N-tails mainly reduces the nucleosome stability. Acetylation in histone H3 globular part on lysine56 regulates the activity of nucleosome by affection of its structure. Nucleosomes with H3K56ac increase the chromatin breathing and cooperate with proteins involved in DNA reparation, replication or specific gene transcription activation. In pluripotent embryonic stem cells (ESC), H3K56ac directly interact with major pluripotency regulator, Oct4. Inhibition of H3K56ac incorporation into nucleosomes suppresses pluripotency and activates cell differentiation. Our goal is to reveal the role of H3K56ac in stem cell pluripotency and differentiation processes and its relationship with initiation and elongation phases of gene transcription. However, the level of H3K56ac in hESC lineage CCTL12 was shown to be dynamic during the cell cycle. Therefore we use double thymidine block to separate cell cycle stages and to determine the amount and distribution of H3K56ac by differential proteomic approaches. The level of H3K56ac grew rapidly during G2 phase in compared to the histone H3 level. Because of the antigen rarity, H3K56ac signals were distributed in cell nucleus as hundreds of well distinguished fluorescent foci. CCTL12 cells showed differential H3K56ac foci level during cell cycle stages in contrast to control, cancer cell lines HeLa and HL-60, where the number of H3K56ac foci raised its maximum in S phase. In conclusion we described cell cycle dependent regulation of H3K56ac nuclear level in embryonic stem cells.