Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase which is composed of two catalytic α- or α'-subunits and two non-catalytic β-subunits. CK2 has been shown to be implicated in embryogenesis, spermatogenesis, and the development of certain organs but its role in basal differentiation processes is only sparsely analyzed. 3T3-L1 cells, which are murine pre-adipocytes, can be induced to differentiate into mature adipocytes within 2 weeks using a combination of insulin, dexamethasone, and isobutylmethylxanthine. We found that the activity of CK2 slightly increases until day 6 and subsequently, decreases in fully differentiated adipocytes. The decrease in activity goes along with a lower expression of all the three subunits of CK2. After inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) or 1,2,5,8-tetrahydroxyanthraquinone (quinalizarin), before day 6, 3T3-L1 cells did not differentiate into adipocytes; inhibition of CK2 after day 6 had no effect on the differentiation process. These results indicated a role of CK2 in early events of the differentiation process and that CK2 is dispensable for late stages of differentiation.