Quidquid agis, prudenter agas et respice finem!—Whatever you do, do it wisely and consider the goal. In consideration of that sage advice, the chicken B-cell line DT40 is an excellent model cell system to study the function of vertebrate genes. In addition to being highly amenable to gene manipulations, the recent influx of genome and gene/protein resources allows for the straightforward selection, design, and targeting of candidate genes for knockout analysis. This chapter will give a step by step standardized protocol to creating a gene knockout mutant in DT40. With careful consideration, the methods and protocols described herein can be easily modified to allow for further gene manipulations such as creating a knockin or a conditional mutant.