Murine embryonic stem (ES) cells are derived from the inner cell mass of 3.5–day-old embryo and have the ability to colonize the germline and form normal gametes following in vitro genetic manipulations. This remarkable characteristic of ES cells has provided the basis for studying normal gene function in the mouse by targeted mutagenesis. Nevertheless, ES cells are very sensitive and need to be manipulated with care for them to retain totipotency after extensive in vitro manipulations. Here we provide straightforward protocols for proper care of these cells. Special emphasis is placed on aspects that are particularly critical for proper culture of this cell type.