Abstract: A culture medium for preparation of feeder cells for embryonic stem cells, which can efficiently establish feeder cells for use in culture of embryonic stem cells including human's from limited donor-derived materials and culture them in a condition of a reduced risk of infection, is provided. Further, a preparation method of feeder cells, which is relatively safe even when subjected to coculture with embryonic stem cells including human's, and the resulting feeder cells therefrom are provided. With the culture medium for preparation of feeder cells for embryonic stem cells comprising at least a serum albumin and insulin in a basal medium, a cell population comprising at least one kind of cells selected from fetal skin fibroblasts, fetal myofibroblasts, fetal lung fibroblasts, fetal epithelial cells, fetal endothelial cells, adult skin fibroblasts, adult lung fibroblasts, adult epithelial cells and endothelial cells which can become feeder cells for embryonic stem cells can be stably proliferated.