Many U.S. medical schools conduct holistic review of applicants to enhance the socioeconomic and experiential diversity of the physician workforce. The authors examined the role of first-generation college-graduate status on U.S. medical school application, acceptance, and matriculation, hypothesizing that first-generation (vs. continuing-generation) college graduates would be less likely to apply and gain acceptance to medical school.Secondary analysis of de-identified data from a retrospective national-cohort study was conducted for individuals who completed the 2001–2006 Association of American Medical Colleges (AAMC) Pre-Medical College Admission Test Questionnaire (PMQ) and the Medical College Admissions Test (MCAT). AAMC provided medical school application, acceptance, and matriculation data through 06/09/2013. Multivariable logistic regression models identified demographic, academic, and experiential variables independently associated with each outcome and differences between first-generation and continuing-generation students. Of 262,813 PMQ respondents, 211,216 (80.4%) MCAT examinees had complete data for analysis and 24.8% self-identified as first-generation college graduates. Of these, 142,847 (67.6%) applied to U.S. MD-degree-granting medical schools, of whom 86,486 (60.5%) were accepted, including 14,708 (17.0%) first-generation graduates; 84,844 (98.1%) acceptees matriculated. Adjusting for all variables, first-generation (vs. continuing-generation) college graduates were less likely to apply (odds ratio [aOR] 0.84; 95% confidence interval [CI], 0.82–0.86) and be accepted (aOR 0.86; 95% CI, 0.83–0.88) to medical school; accepted first-generation college graduates were as likely as their continuing-generation peers to matriculate. Students with (vs. without) paid work experience outside hospitals/labs/clinics were less likely to apply, be accepted, and matriculate into medical school. Increased efforts to mitigate structural socioeconomic vulnerabilities that may prevent first-generation college students from applying to medical school are needed. Expanded use of holistic review admissions practices may help decision makers value the strengths first-generation college graduates and other underrepresented applicants bring to medical educationand the physician workforce.