Mouse embryonal carcinoma (EC) cells, also called teratocarcinoma stem cells, are nonpermissive for polyomavirus growth, whereas differentiated derivatives of the cells are permissive. Mutant viruses capable of growing in EC cells can be isolated. They have genomic alterations within the viral enhancer, which is required for viral gene expression and DNA replication. This viral regulatory region was considered as a potential probe for mouse cell differentiation. The 24-bp-long A element within the enhancer was identified as a minimum element, which also shows a lower activity in EC cells compared with the differentiated cells. Transcription factors PEA1/AP1, PEA2/PEBP2, and PEA3/ETS were identified as A element-binding proteins. All of them are absent in EC cells and induced to be expressed when the cells are differentiated. Although PEBP2 has a weaker transactivation activity compared with other two, it is essential for the enhancer function of the A element. Purification and cDNA cloning revealed that PEBP2 has two subunits, DNA-binding alpha (PEBP2alpha) and non-DNA-binding beta (PEBP2beta). PEBP2alpha was found to be highly homologous to a Drosophila segmentation gene, runt, and a human gene AML1 that was identified as a part of the fusion gene, AML1/ETO (MTG8) generated by t(8;21) chromosome translocation associated with acute myelogenous leukemia (AML). Core-binding factor (CBF), which interacts with a murine retrovirus enhancer, was found to be identical to PEBP2. runt, PEBP2alpha and AML1 are now termed RUNX family, which are involved in cell specification during development. There are three mammalian RUNX genes, RUNX1, RUNX2, and RUNX3. RUNX1 is essential for generation of hematopoietic stem cells and is involved in human leukemia. RUNX2 is essential for skeletal development and has an oncogenic potential. RUNX3 is expressed in wider ranges of tissues and has multiple roles. Among others, RUNX3 is a major tumor suppressor of gastric and many other solid tumors.