This protocol describes the preparation of feeder cells from MEF cells or from the STO mouse fibroblast cell line. The cells are rendered mitotically inactive by treatment with {gamma}-irradiation. The feeder layers can then be used to maintain embryonic stem (ES) cells in the undifferentiated state. - [Read Preparing Feeder Cell Layers from STO or Mouse Embryo Fibroblast (MEF) Cells Protocol]