Using Vitex agnus-castus as the research material, we explored the flower bud differentiation process and its physiological characteristics to provide the basis for flowering regulation technology. The morphological and physiological changes during flower development were studied by combining phenophase observation with the paraffin section and measuring the relevant physiological indexes during flower bud differentiation. The results showed that the flower bud differentiation process can be divided into seven stages: undifferentiated stage, total axis inflorescence primordium differentiation stage, primary axis inflorescence primordium differentiation stage, secondary axis inflorescence primordium differentiation stage, microfloral primordial differentiation stage, early and later floral organ differentiation stage, which are multiple differentiation types in one year. The process of flower bud differentiation in different positions of the same plant and single flowers in the same inflorescence is different. The flower bud differentiation forms at different stages after the first season of flowering and often overlaps. The contents of soluble sugar and soluble protein in leaves and flower buds increased and decreased at different stages of flower bud differentiation. In general, the nutrient content in leaves was higher than that in flower buds to ensure nutrient supply. During flower bud differentiation, the overall levels of IAA, ABA, CTK, and GA3 first increased and then decreased, beneficial to flower bud differentiation. It is believed that the accumulation of a large amount of soluble sugar and protein in flower buds and the high carbon-nitrogen ratio is conducive to the smooth completion of flower bud differentiation of Vitex agnus-castus. Low levels of GA3/ ABA and IAA / CTK were advantageous to inflorescence formation. The ratio of ABA / CTK and ABA / IAA increased during the floral primordium differentiation stage and sepal primordium stage. Petal primordium and stamen primordium differentiation needed higher GA3/ CTK, GA3 / ABA, and GA3 / IAA ratios.