Post-translational modifications (PTMs) to histone proteins constitute a major type of epigenetic mechanism that regulates chromatin structure and gene expression patterns in eukaryotes. In addition to their important roles in standard physiology, disruptions in histone PTM signaling patterns have been suggested to be significant, potentially causative factors in various human diseases such as cancer. As most histone PTM work in the chromatin biology field is accomplished using site-specific antibodies, the quantitative measurement of combinational histone PTMs co-occurring on the same molecule has been unmet. Our objectives include the continued development of mass spectrometry-based proteomics and bioinformatic methods for quantitatively interrogating combinatorial histone PTM patterns, and applying these approaches to investigate histone mediated epigenetics mechanisms behind key areas of health related biological research. Here we will specifically apply our approaches to investigate epigenetic histone PTM signaling during human embryonic stem cell differentiation. Our specific aims are three in number and include identifying changing histone PTMs during stem cell differentiation, characterizing combinatorial histone PTM binding protein complexes that translate these PTM patterns, and determining the role of these combinatorial PTMs in maintaining the pluripotent state or facilitating to a specific lineage We expect that these comprehensive proteomic strategies will continue to generate new tools to study epigenetic histone PTMs and generate novel insights into the mechanism of combinatorial histone PTMs in gene regulation during diverse biological events such as cellular differentiation.