Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.