Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. N/A.