Mary Thompson was born an "army brat," and grew up in three states (Missouri, Kentucky, and New York), West Germany, and the occupied city of West Berlin. She has a B.A. in History, with a minor in Folklore, from Samford University in Birmingham, Alabama, and an M.A. in History from the University of Virginia, where her thesis dealt with the relationship between colonists and Native Americans in Georgia and the Carolinas in the mid-18th century. Before joining the Mount Vernon staff, Ms. Thompson worked as a volunteer at two United States Army museums and as a field researcher on a grant project, sponsored by the National Endowment for the Humanities, to identify practitioners of traditional folk crafts in central Alabama.

Since coming to Mount Vernon in 1980, Mary has filled a number of roles: Historic Interpreter (1980); Curatorial Assistant (1980-1986); Curatorial Registrar (1986-1998); Research Specialist (1998-2008); and Research Historian (2008-present). She is currently responsible for research to support programs in all departments at Mount Vernon, with a primary focus on everyday life on the estate, including domestic routines, foodways, religious practices, slavery, and the enslaved community. She has lectured on a variety of subjects, ranging from family life and private enterprise among the slaves at Mount Vernon, to slave resistance, the diet of the Mount Vernon slaves, Christmas at Mount Vernon, religious practices in the Washington family, and funeral and mourning customs in George Washington's family.

Mary has also authored chapters in a number of books, as well as entries in encyclopedias, and a variety of articles. She curated the travelling exhibition, Treasures from Mount Vernon: George Washington Revealed, which opened in 1998 and travelled to five cities over the next 18 months. Her first book was In the Hands of a Good Providence: Religion in the Life of George Washington (Charlottesville: University of Virginia Press, 2008), for which she received the 2009 Alexandria History Award from the Alexandria [Virginia] Historical Society and the 2013 George Washington Memorial Award from the George Washington Masonic National Memorial. More recently, Applewood Books asked her to write A Short Biography of Martha Washington aimed at the young adult market; it was published in December 2017. She was a major contributor to both The Mount Vernon Ladies' Association: 150 Years of Restoring George Washington's Home and Dining with the Washingtons: Historic Recipes, Entertaining, and Hospitality from Mount Vernon, published by Mount Vernon in 2010 and 2011, respectively. Her book on George Washington, the institution of slavery, and the enslaved community at Mount Vernon, was published by the University of Virginia Press.