PPARgamma and C/EBPalpha cooperate to control preadipocyte differentiation (adipogenesis). However, the factors that regulate PPARgamma and C/EBPalpha expression during adipogenesis remain largely unclear. Here, we show PTIP, a protein that associates with histone H3K4 methyltransferases, regulates PPARgamma and C/EBPalpha expression in mouse embryonic fibroblasts (MEFs) and during preadipocyte differentiation. PTIP deletion in MEFs leads to marked decreases of PPARgamma expression and PPARgamma-stimulated C/EBPalpha expression. Further, PTIP is essential for induction of PPARgamma and C/EBPalpha expression during preadipocyte differentiation. Deletion of PTIP impairs the enrichment of H3K4 trimethylation and RNA polymerase II on PPARgamma and C/EBPalpha promoters. Accordingly, PTIP(-/-) MEFs and preadipocytes all show striking defects in adipogenesis. Rescue of the adipogenesis defect in PTIP(-/-) MEFs requires coexpression of PPARgamma and C/EBPalpha. Finally, deletion of PTIP in brown adipose tissue significantly reduces tissue weight. Thus, by regulating PPARgamma and C/EBPalpha expression, PTIP plays a critical role in adipogenesis.