Stephen L. Johnson was raised in Nashville, Tennessee, the middle (with his twin brother) of four children, growing up in the pre- and post-Civil Rights Era. His father received his degree in electrical engineering and taught in that discipline at Vanderbilt University, though he also pursued a degree in divinity; his mother was a trained psychologist. Johnson partook in the normal activities of childhood, including Boy Scouts and music, but he had a very high affinity for and interest in writing. He matriculated at Vanderbilt University with the intention of becoming a writer. After deciding against becoming a novelist, Johnson's interest in science was piqued while working in Lee Limbird's pharmacology lab, though he still had some trepidation about whether or not science actually suited him. Ultimately he decided to pursue science and was accepted into the genetics department at the University of Washington, Seattle, where he worked under Breck Byers on fusing Cdc4 and LAC-Z genes in yeast. While at Washington he was also fortunate to be mentored by Nobel laureate Leland H. Hartwell.

Upon finishing his graduate studies Johnson decided to remain in the Northwest and began to work on zebrafish with James A. Weston and Charles A. Kimmel at the University of Oregon, Eugene. While there he worked on tissue regeneration mutants, pigment patterns, isometric growth, and genetic mapping, and he developed inbred strains and centromere markers for mapping the zebrafish genome. Johnson then accepted a position at Washington University School of Medicine to continue his work.
Near the end of the interview Johnson uses the topics already discussed in his oral history as a way to reflect upon his scientific development and the ways in which he mentors students and how he thinks about and practices science. The interview concludes with Johnson's thoughts on the role of technological innovation on his work; the advantages and disadvantages of competition in science; the direction of the national science agenda; the National Institutes of Health; gender issues; and the impact of the Pew Scholars Program in the Biomedical Sciences funding on his work.