Mediator is the evolutionarily conserved coactivator required for the integration and recruitment of diverse regulatory signals to basal transcription machinery. To elucidate the functions of metazoan Mediator, we isolated Drosophila melanogaster Med6 mutants. dMed6 is essential for viability and/or proliferation of most cells. dMed6 mutants failed to pupate and died in the third larval instar with severe proliferation defects in imaginal discs and other larval mitotic cells. cDNA microarray, quantitative reverse transcription-PCR, and in situ expression analyses of developmentally regulated genes in dMed6 mutants showed that transcriptional activation of many, but not all, genes was affected. Among the genes found to be affected were some that play a role in cell proliferation and metabolism. Therefore, dMed6 is required in most cells for transcriptional regulation of many genes important for diverse aspects of Drosophila development.