Arsenic trioxide (ATO) synergistically promotes all trans retinoic acid (ATRA)-induced differentiation of PML-RARalpha negative HL-60 myeloblastic leukemia cells. In PML-RARalpha positive myeloid leukemia cells, ATO is known to cause degradation of PML-RARalpha with subsequent induced myeloid differentiation. We found that ATO by itself does not cause differentiation of the PML-RARalpha negative HL-60 cells, but enhances ATRA's capability to cause differentiation. ATO augmented ATRA-induced RAF/MEK/ERK axis signaling, expression of CD11b and p47(PHOX), and inducible oxidative metabolism. ATO enhanced ATRA-induced population growth retardation without evidence of apoptosis or enhanced G1/G0 growth arrest. Compared to ATRA-treated cells, the ATRA plus ATO-treated cells progressed more slowly through the cell cycle as detected by a slower rate of accumulation in G2/M following nocodazole treatment. Hoechst/PI staining showed that low-dose ATO did not induce apoptosis. In summary, our results indicate that ATO in conjunction with ATRA is of potential chemotherapeutic use in PML-RARalpha negative leukemias.