Abstract Acute Promyelocytic Leukemia, (APL, M3 FAB-subtype) is characterized by the chromosomal translocation t(15;17) and the expression of the oncogenic fusion protein PML-RARα (promyelocytic leukemia gene-retinoic acid receptor -α); this AML subtype is strikingly responsive to pharmacological doses of retinoic acid (RA), which can overcome the proliferative and anti-differentiation effects of PML-RARα, thus representing a mainstay of the induction and maintenance therapy of this leukemia. Despite the progress achieved in the last decade in the understanding of the RA-dependent molecular pathways, the discrete regulation of RA receptors function in APL still remains unclear. CK2 is a tetrameric (2 α catalytic and 2 β regulatory subunits) protein kinase overexpressed in a large array of tumors, which has been found to phosphorylate and regulate the activity of key myeloid transcription factors and proteins involved in chromatin remodelling. However, CK2 ability to modulate the myeloid developmental program and the RA-dependent cascade has never been investigated. In this work, we found that CK2 is highly expressed and active in APL cells. Its localization was usually detected in the cytoplasm, with a scant speckled nuclear pattern; upon RA stimulation most of the protein relocalized in perinuclear areas in the cytosol. Moreover, while the protein levels of the β regulatory subunit progressively fell, the levels and activity of the α catalytic subunit, after RA treatment of APL cells, was found consistently elevated. To understand whether CK2 plays a role in RA-induced myeloid differentiation, we employed specific CK2 inhibitors (tetrabromobenzotriazole (TBB)-derivatives K27 and K17) and RNA interference to hamper its function. NB4 and primary APL cells displayed an inhibited terminal differentiation upon RA if CK2 activity was blocked with either one of these means, as evaluated by i) the persistence of an immature morphology, ii) the absence of expression of CD11b and CD11c and iii) the greatly reduced production of superoxide anion at the NBT-reduction assay. All these effects were not caused by an increased apoptosis of APL cells, nor by an inhibition of the reshaping of PML nuclear bodies. Instead, CK2 inhibition affected the RA-dependent G1 arrest of APL cells, a prerequisite for the terminal differentiation program to take place. We have also found that RARα-dependent transcription was greatly impaired by CK2 inhibition, and we have evidence of a physical interaction between these two molecules. Last, to clarify the molecular basis of this unpredicted role of CK2 in myeloid differentiation, we have undertaken a proteomic/phosphoproteomic analysis in response to RA. Surprisingly, CK2 inhibition prevented or attenuated a significant amount of the major phosphorylation changes occurring in response to RA. This study emphasizes the role of CK2 in the differentiation induced by RA in AML. Further studies are needed to identify the CK2 targets involved the RA action.