Pharmacological modulators of metabolism and AMP-dependent kinase (AMPK) inhibit proliferation of tumor cells. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a compound commonly used as an AMPK-modulator, induced AMPK-independent differentiation of U937 cells. Autophagy has been described as an AMPK-independent effect of AICAR in other cells. Therefore, the aim of this study was to determine the role of autophagy and metabolism in differentiation of U937 cells. The results showed that AICAR-mediated effects were not mimicked by specific AMPK agonist and that AICAR had no significant effects on aerobic glycolysis. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II. These effects were not observed in response to metformin, an AMPK agonist without differentiative properties. The increase in LC3B-II was due to the increase in autophagy flux and the autophagy inhibitor 3-methyladenine inhibited differentiation in response to all inducers. The effects of AICAR and ATRA on differentiation markers did not depend on Beclin-1, hVps34 and Atg7. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that differentiation does not depend on the classical or canonical autophagy pathway.