Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.