This study was conducted to improve establishing autologous embryonic stem cells (ESCs) by culture of preantral follicles and parthenogenetic activation of oocytes. First, paternal inheritance of the follicle donor was changed without altering maternal heredity by employing B6CBAF1 instead of B6D2F1 mice. A significant increase in the establishment of parthenogenetic ESCs was detected after the change, and a different gene expression pro-file was detected in the ESCs established. Among 62 stemness-related genes showing different expression level between two strains, 35 (56.5%) were lower in the rarely established ESCs (B6D2F1) than in the easily established ESCs (B6CBAF1). Several paternally expressed genes were aberrantly expressed in the rarely established ESCs. Second, the establishment of parthenogenetic ESCs in B6D2F1 was significantly improved when preantral follicles were cultured in glutathione (GSH)-containing medium. In the ESCs derived from GSH-treated follicles, 77% of the 62 genes showing the difference increased their expression. Translation of several proteins related to stemness (Wnt-1, beta-catenin, p-p44/42, and smad) was similar between the parthenogenetic ESCs established after GSH treatment and the control E14 ESCs. We concluded that change in genetic inheritance and exposure of in vitro-growing ovarian follicles to GSH contributes to improving establishment of parthenogenetic ESCs, which may help increase the feasibility of the established lines for patient-specific, stem cell therapy.