Inducing null mutations by means of homologous recombination provides a powerful technique to investigate gene function and has found wide application in many different fields. However, it was realized some time ago that the specific way in which such knockout mutants are generated can be confounding, making it impossible to separate the effects of the induced null mutation from those of alleles originating from the embryonic stem cell donor. In addition, effects from null mutations can be altered on different genetic backgrounds. Here we present some simple breeding strategies to test for flanking gene effects that are compatible with the recommendations of the Banbury Conference on Genetic Background in Mice and with common practices of creating and maintaining mouse knockout lines.