Lymphatic contractile dysfunction has been identified in several diseases, including lymphedema, yet a detailed molecular understanding of lymphatic muscle physiology has remained elusive. With the advent of genetic methods to manipulate gene expression in mice, a set of new tools became available for the investigation and visualization of the lymphatic vasculature. To gain insight into the molecular regulators of lymphatic contractile function, regulated primarily by the muscle cell layer encircling lymphatic collecting vessels, ex vivo approaches to allow control of hydrostatic and oncotic pressures and flow have been invaluable, complementing in vivo methods. While the original ex vivo techniques were developed for lymphatic vessels from large animals, and later adapted to rat vessels, here we describe modifications that enable the study of isolated, pressurized murine lymphatic collecting vessels. These methods, used in combination with transgenic mice, can be a powerful tool to investigate the molecular and cellular mechanisms of lymphatic function.