In Drosophila, two chromosomes require special mechanisms to balance their transcriptional output to the rest of the genome. These are the male-specific lethal complex targeting the male X chromosome and Painting of fourth targeting chromosome 4. Here, we explore the role of histone H3 methylated at lysine-36 (H3K36) and the associated methyltransferases—Set2, NSD, and Ash1—in these two chromosome-specific systems. We show that the loss of Set2 impairs the MSL complex–mediated dosage compensation; however, the effect is not recapitulated by H3K36 replacement and indicates an alternative target of Set2. Unexpectedly, balanced transcriptional output from the fourth chromosome requires intact H3K36 and depends on the additive functions of NSD and Ash1. We conclude that H3K36 methylation and the associated methyltransferases are important factors to balance transcriptional output of the male X chromosome and the fourth chromosome. Furthermore, our study highlights the pleiotropic effects of these enzymes.