Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys(20)) trimethylation. An increase in H4-Lys(20) trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys(20) is localized to heterochromatin, the increase in H4-Lys(20) trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys(20) trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys(20) is therefore the most suitable substrate for H4-Lys(20) trimethylase. Summarizing, histone H4-Lys(20) hypotrimethylation correlates with H4 hyperacetylation and H4-Lys(20) hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin.