The histone methyltransferases MLL and ASH1L are trithorax-group proteins that interact genetically through undefined molecular mechanisms to regulate developmental and hematopoietic gene expression. Here we show that the lysine 36-dimethyl mark of histone H3 (H3K36me2) written by ASH1L is preferentially bound in vivo by LEDGF, an MLL-associated protein that co-localizes with MLL, ASH1L and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild type MLL proteins to chromatin at key leukemia target genes, and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely KDM2A, an H3K36me2 demethylase and Polycomb-group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies illuminate the molecular mechanisms underlying epigenetic interactions wherein placement, interpretation and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a target for therapeutic intervention. Investigation of multiple transcription factors and histone modification marks in MV4-11 human leukemia cells.