The t(8;21) translocation is the most common cytogenetic abnormality in acute myeloid leukemia (AML). Although t(8;21) AML patients have a relatively favorable prognosis, relapse is a frequent occurrence, underscoring the need to develop novel therapeutic approaches. Here, we showed that t(8;21) AML is characterized by frequent mutation and overexpression of CCND2 . Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for the selective CDK4/6 inhibitors palbociclib and abemaciclib than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. CDK4/6 inhibition decreased MAP-ERK and PI3K-AKT-mTOR signaling pathway activity, induced LC3B-I to LC3B-II conversion, and enhanced autophagosome formation, suggesting autophagy induction. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with the CDK4/6 inhibitor abemaciclib significantly increased the percentage of apoptotic (Annexin V positive) cells, whereas CQ or LY294002 single treatment had no significant effects. The effectiveness of co-inhibiting CDK4/6 and autophagy was confirmed in primary t(8;21) AML cells. The results suggest that the combination of CDK4/6 and autophagy inhibitors had a synergistic effect on inducing apoptosis, suggesting a novel therapeutic approach for the treatment of t(8;21) AML.