Gene targeting is a powerful tool to dissect gene’s functions. Recent developments of recombineering and sitespecific recombination have dramatically increased the efficacy of gene targeting. In last decade there has been a huge influx of data from gene targeting approaches to airway diseases. The approaches greatly help us to understand the mechanisms from signal transductions to immune response in airway diseases. Recent human association studies revealed many novel genes underlying complicated airway diseases. The next challenge will be the systematic analysis of the precise functions of these genes in the pathogenesis of the diseases. This review looks into the current development of gene targeting in mice in the investigations of airway diseases. It also provides a brief protocol for how to rapidly construct the targeting vectors with recombineering and site-specific recombination systems.