All-trans retinoic acid (ATRA) induces granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL) cells, although the detailed mechanisms are not fully understood. We investigated ATRA-induced cellular responses mediated by the transcription factor FOXO3A in APL cells. FOXO3A was constitutively phosphorylated and localized in the cytoplasm in both APL-derived NB4 cells and primary APL cells. Upon treating the cells with ATRA, FOXO3A phosphorylation was reduced and FOXO3A translocated into the nucleus. In addition, the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a target molecule for FOXO3A, was increased at the transcriptional and protein levels. As expected, transfection of a short hairpin RNA (shRNA) oligonucleotide specific for FOXO3A significantly inhibited ATRA-induced granulocytic differentiation and apoptosis in NB4 cells. In NB4-derived ATRA-resistant NB4/RA cells, neither FOXO3A nuclear localization nor subsequent TRAIL induction was observed after ATRA treatment. Furthermore, forced expression of active FOXO3A in the nucleus induced TRAIL production and apoptosis in NB4/RA cells. We conclude that activation of FOXO3A is an essential event for ATRA-induced cellular responses in NB4 cells. FOXO3A is a promising target for therapeutic approaches to overcome ATRA resistance in APL.