This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate‐mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion‐containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be “pseudotyped” with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV‐G‐pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.