Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukemias (APL) by triggering differentiation of neoplastic cells. Differentiation is mediated by the APL-specific transforming protein PML/RARα and involves its activity as ligand-dependent enhancer factor on RA-target genes. We report here the identification of p21 as a transcriptional target of PML/RARα during RA-induced differentiation of APL cells. We found that RA-treated APL cells undergo two rounds of cell division before entering post mitotic G1, that progression through the G1-S is indispensable for differentiation and coincides with the duration of commitment. RA-treatment induced two peaks of p21 synthesis: early (from the 2nd to the 6th hour), dependent on PML/RARα expression and associated with G1-S transition and high CDK activity; late (from 3rd to the 4th day), independent from PML/RARα and associated with G1 block and low CDK activity. Increased p21 in PML/RARα cells during G1-S had no effect on the cell cycle while an antisense p21 prevented RA-induced differentiation without altering G1-S transition and the late G1 block. These results demonstrate that p21 is an effector of the activity of PML/RARα on differentiation and suggest that p21 exerts a function in G1-S connected to differentiation-commitment and uncoupled from cell cycle and CDK inhibition.