Abstract: Improving the characteristics of cotton flower bud differentiation is important for coordinating the relationship between vegetative growth and reproductive growth in cotton plants, building a good cotton population structure, and increasing yield. To explore the regulatory effects and physiological mechanisms of the water supply and planting densities on cotton seedling flower bud differentiation, this study employed two split-plot designed experiments in 2019 on the Qingyuan Experimental Plot of Hebei Agricultural University. The main plot had two water treatmentsfull irrigation (CK) and drought (D), and the sub-plot contained three density treatments6×10⁴ plants·hm^-2 (low density), 9×10⁴ plants·hm^-2(medium density), and 12×10⁴ plants·hm^-2 (high density). By using 'Nongda 601' as the experiment material, the effects of different water supplies and planting densities on floral bud differentiation stage and progression, and the related physiological indexes (hormone, soluble sugar, and soluble protein contents) of cotton seedlings were studied. Before cotton sowing, different amounts of soil moisture were set: the control treatment was 900 m³·hm^-2, and the drought treatment was 450 m³·hm^-2. The results of microstructure showed that: 1) The differentiation speed of cotton shoot apical meristem accelerated under drought conditions. Under high-density conditions, the aspect ratio of cotton shoot apical meristem decreased, which slowed the differentiation rate. Under the same planting density, the position of the first fruit branch lowered down, but under the same water treatment, the position of the first fruit branch significantly raised with increased planting density (P < 0.05). Therefore, the interaction effect of the two factors is significant. 2) The drought and density treatments significantly impacted the soluble protein, soluble sugar, and endogenous hormone contents of the cotton shoot apical meristem. At the 2-leaf stage, the soluble protein and soluble sugar contents of the low-density treatment were significantly higher than those of the other two treatments, and the contents of endogenous hormone gibberellin (GA₃)was higher. The cytokinin (ZR) content was also lower. The ratios between endogenous hormones of indole-3-acetic acid/abscisic acid (IAA/ABA), ZR/ABA, and (IAA+ZR+GA₃)/ABA increased with increasing plant density. With flower bud differentiation, there was no significant difference in the contents of soluble protein and soluble sugar among the treatments. However, the ratios of IAA/ABA, ZR/ABA, and (IAA+ZR+GA₃)/ABA in the high-density treatment were significantly lower than those in other treatments (P < 0.05). The GA₃/ABA ratio was higher than that in other treatments. 3) Principal component analysis of the indexes of flower bud differentiation showed that the contents of soluble sugar and ZR at the shoot apical meristem had the greatest influence on initial flower bud differentiation at the 2-leaf and 3-leaf stages. Therefore, under drought conditions, the shoot apical meristem of cotton at a density of 6×10⁴ plants·hm^-2 differentiated faster, the nutrient substance and GA₃ content were higher, and the ZR content was lower, all of which was conducive to the initiation of cotton flower bud differentiation. However, the 12×10⁴ plants·hm^-2 treatment reduced the aspect ratio of the shoot apical meristem, increased the GA₃/ABA ratio, and slowed flower bud differentiation. The results provide a theoretical basis for the regulation of flower bud differentiation in cotton.