Gene targeting, which allows the efficient modification of specific murine genes, plays important roles in elucidating physiological function of genes in vivo, dissecting the genetic mechanisms of human disease and identifying potential targets for drug development. In recent years, the development of conditional gene targeting has provided novel oppor-tunities for gene inactivation in specific cells or organs, and at specific time points, both during development and in the adult animals. In this review, we introduce the history of gene targeting and recent progress of gene targeting technology and its applications in other model organisms.