AML1 (Acute Myeloid Leukemia 1; also known as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays important roles in initiating definitive hematopoiesis, thymocyte development, and platelet production. AML1’s DNA-binding is mediated through its Runt domain, and their further association with the non-DNA-binding subunit, CBFβ stabilizes the resultant ternary complex to be functional. AML1 is frequently involved in leukemia-associated chromosome translocations, through which AML1 fusion-proteins with strong dominant-negative effects to normal CBF are generated, thus contributing to the leukemic transformation. Recently, genomic mutations of the AML1 gene locus have been described to associate with cases of acute myeloblastic leukemia and myelodysplastic syndromes (MDS), and pedigrees of familial platelet disorder with propensity to develop acute myeloblastic leukemia (FPD/AML). Frequent sites of the point-mutations are confined to the residues within the Runt domain, such as, R139, R174, or R177, which are important for the DNA-contact of the molecule. Preliminary results from studies using knockin mice carrying mis-sense mutations for these residues so far revealed that the mutants are biologically inactive when homozygously inherited. However, the mechanisms how the mutations contribute to the onset of the leukemia remain largely to be elucidated. In order to further characterize the aberration of AML1, we generated another genome-modified mouse line which harbors an insertion mutation of this gene locus. As reported previously, we identified a case of refractory anemia with excess of blasts (RAEB) whose blast cells have a mutated allele where three base-pair, ATC, is inserted after codon 150 (I150ins) of the molecules. By this mutation, an additional isoleucine residue is inserted into the 10th β-strand of the Runt domain sequences which provides a facet to interact with CBFβ. We introduced the cDNA of I150ins into AML1/Runx1 gene locus by means of a gene-knockin approach in mouse ES cells, and transmitted the allele into mouse germline through conventional methods. Subsequent heterozygous mice were viable and fertile. In contrast, homozygous mice die in utero similarly to the mice with simple disruption of this gene. These results indicate that this insertion mutation leads to the complete loss of its biologic function as were observed for the point-mutant mice. Further careful examinations of the heterozygous mice for this mutant allele in comparison with those for point-mutation animals and simple haplo-insufficient ones will provide valuable insights into the molecular mechanisms of AML1-related leukemic transformation.