Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified. By adding Glivec 10(-5) M, a tyrosine kinase receptor c-kit inhibitor, only during EB formation, we for the first time succeeded in suppressing in vitro formation of ICC in the ES gut. The ES gut without ICC did not exhibit any movements. However, it appeared that Glivec 10(-6)-10(-7) M rather increased number of ES guts with spontaneous movements associated with increase of intracellular Ca2+ concentration ([Ca2+](i)). These results suggest ICC is critical for in vitro formation of ES guts with spontaneous movements. (c) 2007 Elsevier Inc. All rights reserved.