Stein attended Radcliffe College, then an annex of Harvard University, from 1893 to 1897 and was a student of psychologist William James. With James's supervision, Stein and another student, Leon Mendez Solomons, performed experiments on normal motor automatism, a phenomenon hypothesized to occur in people when their attention is divided between two simultaneous intelligent activities such as writing and speaking.
These experiments yielded examples of writing that appeared to represent "stream of consciousness", a psychological theory often attributed to James and the style of modernist authors Virginia Woolf and James Joyce. In 1934, behavioral psychologist B.F. Skinner interpreted Stein's difficult poem Tender Buttons as an example of normal motor automatism. In a letter Stein wrote during the 1930s, she explained that she never accepted the theory of automatic writing: "[T]here can be automatic movements, but not automatic writing. Writing for the normal person is too complicated an activity to be indulged in automatically."
At Radcliffe, she began a lifelong friendship with Mabel Foote Weeks, whose correspondence traces much of the progression of Stein's life. In 1897, Stein spent the summer in Woods Hole, Massachusetts, studying embryology at the Marine Biological Laboratory.
William James, who had become a committed mentor to Stein at Radcliffe, recognizing her intellectual potential, and declaring her his "most brilliant woman student", encouraged Stein to enroll in medical school, although Stein professed she had no interest in either the theory or practice of medicine. She spent two years at Johns Hopkins Medical School, failing two courses and leaving without a degree. Ultimately, medical school had bored Stein, and she had spent many of her evenings not applying herself to her studies, but taking long walks and attending the opera.
Stein's tenure at Johns Hopkins was marked by challenge and stress. Men dominated the medical field, and the inclusion of women in the profession was not unreservedly or unanimously welcomed. Writing of this period in her life ("Things As They Are", 1903) Stein often revealed herself as a depressed young woman dealing with a paternalistic culture, struggling to find her own identity, which she realized could not conform to the conventional female role. Her uncorseted physical appearance and eccentric mode of dress aroused comment and she was described as "Big and floppy and sandaled and not caring a damn".
Asked to give a lecture to a group of Baltimore women in 1899, Stein gave a controversial speech titled "The Value of College Education for Women", undoubtedly designed to provoke the largely middle-class audience. In the lecture Stein maintained:
"average middle class woman [supported by] some male relative, a husband or father or brother,...[is] not worth her keep economically considered." [This economic dependence caused her to become] oversexed...adapting herself to the abnormal sex desire of the male...and becoming a creature that should have been first a human being and then a woman into one that is a woman first and always."—
While a student at Johns Hopkins and purportedly still naïve about sexual matters, Stein experienced an awakening of her latent sexuality. Sometime in 1899 or 1900, she became infatuated with Mary Bookstaver who was involved in a relationship with a medical student, Mabel Haynes. Witnessing the relationship between the two women served for Stein as her "erotic awakening". The unhappy love triangle demoralized Stein, arguably contributing to her decision to abandon her medical studies.
In 1902 Stein's brother Leo Stein left for London, and Stein followed. The following year the two relocated to Paris, where Leo hoped to pursue an art career.