Traditionally, knockout experiments are performed in ES cells derived from the 129 mouse strain, followed by backcrossing with the more robust C57BL/6 strain. C57BL/6-derived ES cells have only occasionally been used in this process. We compared C57BL/6- with 129-derived ES cells directly and reviewed the literature. We found that, although some steps are less efficient, the advantages of C57BL/6 mice more than compensate for these drawbacks.