The combination of fludarabine, cytarabine (ara-C) and G-CSF (FLAG) is routinely used in the treatment of acute myeloid leukemia (AML). In this study we characterized the interactions between fludarabine, ara-C and G-CSF in vitro using AML blasts. Exposure to G-CSF alone resulted in a higher leukemic cell survival (LCS), which might be indicative of increased proliferation. The LCS decreased significantly from 69.7 to 54.0% when blasts were exposed to G-CSF 21 h prior to incubation with ara-C (p=0.01). In contrast, LCS increased significantly (from 55.6 to 69.0%; p=0.04) after sequential exposure to G-CSF and fludarabine. Exposure to 4 combinations of fludarabine (4 h; 0.14 microM and 0.55 microM) and ara-C (96 h; 0.21 and 0.82 microM) (FLA) resulted in additive cytotoxicity. The triple combination (FLAG), 21 h 5 microM G-CSF followed by 4 h fludarabine (0.14 and 0.55 microM) and finally ara-C (0.21 and 0.82 microM) for 96 h also resulted in an additive cell kill. In conclusion, these data support the clinical use of G-CSF in combination with ara-C, and the combination of ara-C and FLA. Pre-exposure to G-CSF before FLA (FLAG) did not result in increased cytotoxicity in our experiments, indicative of similar anti-leukemic activity.