The honey bee queen plays a central role in the Apis mellifera L. (Hymenoptera: Apidae) colony, and her high reproductive capacity is fundamental for building up the workforce of a colony. Caste development in honey bee females involves elaborate physiological pathways unleashed at the beginning of the first larval instars, with juvenile hormone (JH) playing a crucial role. Here we took advantage of established in vitro rearing techniques to conduct a 2 × 2 experimental design and test initial rearing age (young vs old) and JH treatment (JH III vs solvent control) to enlighten the role of nutrient quality and JH in shaping honey bee female fertility, morphological features related to queenliness, and key physiological parameters (hemolymph vitellogenin/Vg, sugar levels, and Vg transcript levels). Our results show that while the age at initial larval rearing had major impacts on external morphology development, where younger larvae exhibited a higher probability to develop into queen-like adults morphotypes, the JH application during the larval stage improved physiological pathways related to ovary development and metabolism during the ontogenic development. We detected that the supplementation of queen larvae with JH promoted important benefits regarding queen fertility as the increase of ovariole number and vg levels at hemolymph, both crucial factors at eggs production. The data presented here provide guidance in efforts to improve honey bee queen quality, especially in light of frequent episodes of queen failures in the beekeeping industry.