The effects of End-of-Day (EOD)-cooling on floral differentiation and cut flower characteristics in carnation were verified using both short-term model-based experiments in a growth chamber and long-term demonstrative experiments in a greenhouse. In the model experiment, flower buds were undifferentiated until 43 days after pinching in both control and End-of-Night-cooled plants. In contrast, in the EOD-cooling treatment, every set reached the reproductive phase by 22 days after treatment. At the same node position, a three-stage advancement in floral differentiation was observed after EOD-cooling, but not after the other two treatments. This suggested that EOD-cooling promoted both floral differentiation and development in carnation. For the demonstrative experiment, significant differences between EOD-cooled and control plants varied each year in days to flowering, node order of flowering, and stem weeping angle. Because environmental factors could affect variation, correlations between the diurnal temperature range and the difference in each survey component were examined using regression analyses. Moderate (days to flowering) and a strong (node order of flowering) correlations were found with the diurnal temperature range. Thus, the EOD-cooling treatment was advantageous in suppressing delayed flowering over a large diurnal temperature range during floral differentiation. The difference in stem weeping angle showed a strong correlation with the difference in minimum temperature during the stem elongation stage. Improving the stem quality by EOD-cooling tended to be effective under high minimum-temperature conditions.