Methylation on K4 of H3 (H3K4me) is one of the prominent histone modification marks that correlates strongly with active transcription in yeast and metazoans. Accumulating studies in metazoans have implicated misregulation of H3K4 methylation in the pathogenesis of cancer and in developmental defects, further emphasizing the importance of understanding the regulation of H3K4 methylation. Genetic and cell-based studies have documented a crosstalk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little, if any, direct biochemical evidence exists explaining the mechanism underlying H2Bub-dependent H3K4 methylation on chromatin templates. By taking advantage of an in vitro histone methyltransferarse assay employing a reconstituted human H3K4 methyltransferase complexes and a recombinant chromatin template containing fully ubiquitylated H2B, we demonstrate that only subset of human H3K4 methyltransferase complexes exhibit H2Bub-dependent H3K4 methylation activity. These studies establish minimal components of human H3K4 methyltransferase complexes required for H2Bub-dependent H3K4 methylation and also provide a mechanistic basis for H2Bub-dependent H3K4 methylation.