Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.