To identify potent bioactive factors for in vivo tissue regeneration by comprehensive screening remains a challenge for regenerative medicine. Here we report the development of an ES cell‐based monitoring system for osteogenic differentiation, the identification of a potent combination of osteogenic genes using such a system, and an evaluation of its therapeutic potentials. ES cells were isolated from mice carrying a transgene expressing GFP driven by the 2.3 kb fragment of rat type I collagen α(1) promoter. Using these cells engineered to fluoresce on osteogenic differenti‐ation, we screened cDNA libraries and combinations of major osteogenesis‐related genes. Among them, the combination of constitutively active activin receptorlike kinase 6 (caALK6) and runt‐related transcription factor 2 (Runx2) was the minimal unit that induced fluorescence. The combination efficiently induced os‐teogenic differentiation in various cell types, including terminally differentiated nonosteogenic cells. The cooperative action of the combination occurred through protein stabilization of core binding factor beta (Cbfb), induction of Runx2‐Cbfb complex formation, and its DNA binding. Furthermore, transplantation of a mono‐layer sheet of fibroblasts transduced with the combination achieved bone regeneration within 4 wk in mouse calvarial bone defects. Thus, we successfully identified the potent combination of genes for bone regeneration, which helped broaden cell sources.—Ohba, S., Ikeda, T., Kugimiya, F., Yano, F., Lichtler, A. C., Nakamura, K., Takato, T., Kawaguchi, H., Chung, U. I. Identification of a potent combination of osteogenic genes for bone regeneration using embryonic stem (ES) cell‐based sensor. FASEB J. 21, 1777–1787 (2007)