To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPβ and/or C/EBPδ by gene targeting. Approximately 85% of C/EBPβ (−/−)· δ (−/−) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPβ (−/−)· δ (−/−) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPβ (−/−)· δ (−/−) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPβ (−/−)· δ (−/−) mice exhibit normal expression of C/EBPα and PPARγ, despite impaired adipogenesis. These results demonstrated that C/EBPβ and C/EBPδ have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPα and PPARγ does not always require C/EBPβ and C/EBPδ, but co-expression of C/EBPα and PPARγ is not sufficient for complete adipocyte differentiation in the absence of C/EBPβ and C/EBPδ.