The European flat oyster (Ostrea edulis) is a species of significant ecological, commercial and cultural importance. Interest in scaling up its production is growing rapidly, however, challenges in managing reproduction in culture remain. This study aimed to improve understanding of reproductive control in female broodstock by analysing the gene expression of the key endocrine factors APGWamide, GnRH (gonadotropin releasing hormone) and GPB5 in the visceral ganglion, as well as vitellogenin in the gonads. This was performed alongside histological classification of individuals by sex and reproductive stage. Oysters were subjected to three different conditioning regimes: a reference regime consisting of ambient temperature and a long-day photoperiod of 18-h light: 6-h dark (REF(Amb/18:6)), and separately, two constant temperature regimes of 14 °C, either under simulated natural photoperiod (14C/NP) or constant 24-h light (14C/LL). Histology revealed a female-to-male shift across regimes. Gametogenesis progressed under all conditions, but females under constant light (14C/LL) showed less uniform development. Apgwamide expression significantly increased as females developed across all regimes. Vitellogenin expression also significantly increased over time and with gametogenesis under the REF(Amb/18:6) and 14C/NP regimes. No clear patterns were observed for gnrh and gpb5 expression in this dataset, though their roles in reproduction cannot be excluded. Altogether, these findings support APGWamide and vitellogenin as key factors of interest in female reproduction, with light, as well as the known effects of temperature, influencing development. This research provides insights to support broodstock conditioning and European flat oyster production and restoration efforts.