Introduction: The high number of mutations observed in many cancers including acute myeloid leukemia (AML) led to the mutator phenotype hypothesis, which suggests that an early initiating mutational event acts as a driver for the acquisition of additional mutations which eventually lead to transformation of normal cells to the malignant pheno- type. The RUNX1/ETO fusion gene, a product of t(8;21) translocation, is one of the most common initiating genetic lesion observed in AML. The purpose of the study was to design and establish a reliable flow cytometric method to determine whether the RUNX1/ETO fusion oncoprotein predisposes cells to mutations in leukemia-relevant genes such as NPM1. Methods: TK6 human lymphoblastoid cells and its derivative RUNX1/ETO-positive cell clones were used to evaluate the effect of RUNX1/ETO expression on mutation frequency in NPM1 gene. A flow cytometric meth- od was established using commercially available NPM1 mutant-specific polyclonal antibody to detect the mutated NPM1 event in a cell population. Results: Analysis showed that RUNX1/ETO significantly increase the type A exon 12 NPM1 mutation frequency over time. Furthermore, the level of RUNX1/ETO expression was observed to influ- ence the induced mutation frequency where TK6 clone with high level of RUNX1/ETO expression had a significant higher NPM1 mutation frequency compared to TK6 clone that has lower RUNX1/ETO expression. Conclusion: The data from this study has demonstrated that RUNX1/ETO expression predisposes cells to the acquisition of the type A exon 12 NPM1 mutation, suggesting that this fusion protein drives genomic instability and mutagenesis at loci relevant to leukemogenesis.