PU.1 transcription factor is a critical regulator of hematopoiesis and leukemogenesis. Because PU.1 interacts with transcription factors GATA-2 and C/EBPalpha, and both are involved in the regulation of adipogenesis, we investigated whether PU.1 plays a role in the regulation of adipocyte differentiation. Our data indicate that PU.1 is expressed in white adipose tissue. PU.1 protein can also be detected in cultured 3T3-L1 adipocytes. Forced expression of PU.1 in 3T3-L1 cells inhibits adipocyte differentiation, whereas deletion of the transactivation domain of PU.1 abolishes this effect. The inhibition of adipocyte differentiation by PU.1 is achieved, at least in part, through repression of the transcriptional activity of C/EBPalpha and C/EBPbeta. Furthermore, GATA-2 and PU.1 have an additive inhibitory effect on C/EBP transactivation and adipogenesis. Finally, the expression of PU.1 is increased in white adipose of obese mice.