SignificanceRecurrent histone H3K36M mutations are found in multiple cancer types yet their oncogenic mechanisms remain incompletely understood. Biochemically, H3K36M oncohistone dominantly inhibits several H3K36-specific methyltransferases such as NSD1/2 and SETD2, resulting in decreases in all methylation states of H3K36. We report here that genetic ablation of NSD1 and NSD2, methyltransferases specific for H3K36 dimethylation (H3K36me2), is sufficient to recapitulate H3K36M’s effects on enhancer activation, gene expression, differentiation blockade, and drug sensitivity. Our results suggest that depletion of H3K36me2 represents a key event downstream of the H3K36M mutation and also exposes potential therapeutic vulnerability of H3K36M-mutant tumor cells.