Gene modification technologies play a vital role in the study of biological systems and pathways. Although there is widespread and beneficial use of genetic mouse models, potential shortcomings of gene targeting technology exist, and are not always taken into consideration. Oversights associated with the technology can lead to misinterpretation of results; for example, ablation of a gene of interest can appear to cause an observed phenotype when, in fact, residual embryonic stem cell-derived genetic material in the genetic background or in the area immediately surrounding the ablated gene is actually responsible. The purpose of this review is to remind researchers, regardless of scientific discipline, that the background genetics of a knockout strain can have a profound influence on any observed phenotype. It is important that this issue be appropriately addressed during data collection and interpretation.