When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G₁/S checkpoint synchronously as evidenced by the expression/activation of cdk2-cyclin-E/A, turnover of p27/kip1, hyperphosphorylation of Rb, translocation of cyclin D₁ from nuclei to cytoplasm and GSK-3β from cytoplasm to nuclei, and incorporation of [³H]thymidine into DNA. As the cells cross the G₁/S checkpoint, C/EBPβ acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.