Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34+ versus mature CD34- AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.