An unprecedented influx of funds and support through large programs such as the Global Fund for AIDS, Malaria and Tuberculosis and the World Health Organization's and President's Emergency Plan for AIDS Relief (PEPFAR) has made it possible for more than 1 million persons in resource-limited settings to access AIDS treatment and several million more to be in care and prevention programs. Nevertheless, there remain major challenges that prevent AIDS drugs and care from reaching many more in need, especially in rural settings. The roll-out of a high-quality treatment, care, and prevention program depends on an effective and reliable laboratory infrastructure. This article presents a strategy used by the Institute of Human Virology (IHV)-University of Maryland and its affiliate IHV-Nigeria to establish a multifaceted, integrated tier laboratory program to support a PEPFAR-funded scale-up of its AIDS Care Treatment in Nigeria program, in collaboration with the Centers for Disease Control and Prevention and the Nigerian government, as a possible model for overcoming a key challenge that faces several resource-limited countries trying to roll out and scale-up their HIV/AIDS treatment, care, and prevention program.