Adipocyte differentiation is a complex process triggered and facilitated by transcription factors such as peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein (C/EBP) alpha. Most about the cascade underlying the differentiation process, especially events in the early stages, remain to be elucidated. Early on in adipocyte differentiation, the C/EBPbeta and C/EBPdelta genes are rapidly induced to express and later activate PPARgamma and C/EBPalpha expression. C/EBPbeta also plays a crucial role in mitotic clonal expansion (MCE), the approximately two rounds of mitosis which occurs soon after preadipocytes are stimulated by differentiation inducers and a necessary step for adipocyte differentiation. However, the effect of C/EBPdelta, another member of the C/EBP family, on MCE remains unclear. In the present study, we investigated the role of C/EBPdelta in the early stages of adipogenesis. A remarkable induction of C/EBPdelta gene expression after the initiation of differentiation was observed not in proliferating preadipocytes, but in growth-arrested, differentiable cells. RNAi-mediated knockdown of C/EBPdelta dramatically suppressed cell growth after differentiation was induced, and inhibited conversion into lipid-laden adipocytes. Furthermore, silencing of C/EBPdelta impaired the expression of factor for adipocyte differentiation (fad) 49, which is up-regulated and plays a crucial role early in adipogenesis. Taken together, these findings show that C/EBPdelta is involved in MCE and gene expression in the early stages of adipocyte differentiation.