Upon induction of differentiation, growth-arrested (G(1) phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-beta (C/EBPbeta), initiating a transcriptional cascade. C/EBPbeta immediately undergoes a priming phosphorylation (on Thr(188)) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBPbeta is delayed until further phosphorylation (on Ser(184) or Thr(179)) by GSK3beta occurs. Phosphorylation by glycogen synthase kinase-3beta (GSK3beta) induces S phase entry and thereby mitotic clonal expansion (MCE), a requirement for terminal differentiation. Because MAPK activity is down-regulated before S phase is completed, we sought to identify the kinase that maintains C/EBPbeta in the primed phosphorylated state throughout S phase and MCE. We show here that cdk2/cyclinA, whose expression is activated at the onset of S phase, functions in this capacity. Ex vivo and in vitro experiments show that cdk2/cyclinA catalyzes this delayed priming phosphorylation. Mass spectrometric analysis revealed that cdk2/cyclinA phosphorylates C/EBPbeta on Thr(188) and is required for phosphorylation (on Ser(184) or Thr(179)) of C/EBPbeta by GSK3beta and maintenance of DNA binding activity. Suppression of cdk2 activity by RNA interference or pharmacologic inhibitor disrupts subsequent events in the differentiation program. Thus, MAPK and cdk2/cyclinA act sequentially to maintain Thr(188) of C/EBPbeta in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.