Haldan Keffer Hartline was born on December 22, 1903, in Bloomsburg, Pennsylvania, to Daniel Schollenberger Hartline and Harriet Franklin Hartline. He attended college at Lafayette College in Easton, Pennsylvania, graduating with a B.S. in 1923. He went on to study retinal electrophysiology as a graduate student at Johns Hopkins University, obtaining his M.D. in 1927. Hartline spent the next two years at Johns Hopkins University as a National Research Council fellow in medical sciences.

Between 1929 and 1931, Hartline was a Johnson Traveling Research Scholar from the University of Pennsylvania to the universities of Leipzig and Munich. He travelled extensively in Germany during those years before returning to the United States, where he joined the Eldridge Reeves Johnson Research Foundation for Medical Physics as an assistant professor of biophysics at the University of Pennsylvania. Hartline married Mary Elizabeth Kraus on April 11, 1936. They had three sons: Daniel Keffer, Peter Haldan, and Frederick Flanders...