Direct intrathymic injection is a common procedure used in several types of experimental protocols in the mouse. Currently available approaches involve major surgical procedures that expose the thoracic cavity, resulting in an increased risk of poor recovery and postsurgical complications. The authors sought to refine this surgery to reduce animal pain and distress without compromising overall efficiency of the technique. Using a minimally invasive method that does not expose the thoracic cavity, the authors gave accurately placed intrathymic injections, as confirmed by analyses with a reporter dye. They describe this new approach for intrathymic injection in mice that reduces complications associated with lengthy periods of anesthesia and thoracic cavity exposure.