Activating signal cointegrator-2 (ASC-2), a transcriptional coactivator of multiple transcription factors that include the adipogenic factors peroxisome proliferator-activated receptor gamma (PPARgamma) and C/EBPalpha, is associated with histone H3-Lys-4-methyltransferase (H3K4MT) MLL3 or its paralogue MLL4 in a complex named ASCOM (ASC-2 complex). Indeed, ASC-2-null mouse embryonic fibroblasts (MEFs) have been demonstrated to be refractory to PPARgamma-stimulated adipogenesis and fail to express the PPARgamma-responsive adipogenic marker gene aP2. However, the specific roles for MLL3 and MLL4 in adipogenesis remain undefined. Here, we provide evidence that MLL3 plays crucial roles in adipogenesis. First, MLL3(Delta/Delta) mice expressing a H3K4MT-inactivated mutant of MLL3 have significantly less white fat. Second, MLL3(Delta/Delta) MEFs are mildly but consistently less responsive to inducers of adipogenesis than WT MEFs. Third, ASC-2, MLL3, and MLL4 are recruited to the PPARgamma-activated aP2 gene during adipogenesis, and PPARgamma is shown to interact directly with the purified ASCOM. Moreover, although H3K4 methylation of aP2 is readily induced in WT MEFs, it is not induced in ASC-2(-/-) MEFs and only partially induced in MLL3(Delta/Delta) MEFs. These results suggest that ASCOM-MLL3 and ASCOM-MLL4 likely function as crucial but redundant H3K4MT complexes for PPARgamma-dependent adipogenesis.