Abstract: Objective: This study investigates the effects of cumulus cells and glutathione on the efficiency of in vitro fertilization (IVF) and subsequent development of mouse zygotes to the two-cell stage. Methods: Oocytes were collected from 4–5-week-old female mice following hormone-induced ovulation (PMSG and hCG). Oocytes were isolated in M2 medium, and fresh sperm was isolated in TYH + MBCD medium. The experimental samples were divided into four groups to assess IVF efficiency: 1) Oocytes with intact cumulus cell complexes in mHTF medium without glutathione (GSH); 2) Oocytes with intact cumulus cell complexes in mHTF medium supplemented with GSH; 3) Denuded oocytes (without cumulus cells) in mHTF medium without GSH; 4) Denuded oocytes (without cumulus cells) in mHTF medium supplemented with GSH. Results and Discussion: The percentage of zygotes reaching the two-cell embryo stage 24 h after IVF was 72% in the group with intact cumulus cells, remaining unchanged upon addition of 0.25 mM glutathione. However, in oocytes denuded of cumulus cells using hyaluronidase, a significant difference was observed: supplementation with 0.25 mM GSH increased the rate of two-cell embryos from 32 to 70%, whereas without GSH, only about one-third of zygotes reached this stage. Conclusions: Cumulus cells significantly enhance IVF efficiency and the developmental potential of fertilized mouse oocytes. The addition of glutathione to the IVF medium is essential for successful fertilization when oocytes are removed from their cumulus cell environment.