To realize their potentials, embryonic stem (ES) cells must be maintained in optimal culture conditions that preserve their pluripotency and self-renewal capacity. Mouse embryonic fibroblasts (MEFs) are used to prepare a feeder cell layer that supports the growth of ES cells and the quality of feeders is crucial for the maintenance of undifferentiated ES cells in prolonged culture. The protocols provided in this unit describe aspects of isolation and expansion of MEFs and maintenance of stablished feeder cells. Preparation of mitotically inactivated feeder cell layer (treatment with mitomycin C or I?-irradiation) is also described. In addition, a method for counting cell numbers and a simple method for detection of mycoplasma contamination by in situ DNA staining are also provided. Methodology described has been tested in a real laboratory environment and provides detailed information regarding resource and time requirements as well as critical parameters and troubleshooting.