Environmental factors that influence flight activity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) may have implications for Huanglongbing spread and management. In this work, four studies were conducted to evaluate the effect of environmental conditions on D. citri take-off. In the first, insects were transferred to sweet orange seedlings and confined inside an acrylic cage to verify the take-off periodicity and the effect of environmental factors on this process. In the second, take-off temperature threshold was estimated by recording the number of insects that initiated flight from a platform when subjected to gradual temperature increases from 15 to 39°C. In the other studies, we evaluated the effect of different photoperiods and temperature regimes (third study) and of constant temperatures (fourth study) on the propensity for D. citri flight. Insects were confined in clear plastic bottle cages with tubes of 50 ml placed on the cab, to collect emerged adults that initiated flights. Results showed that a small portion of the tested population (maximum 10%) tends to take off from plants and this behaviour is more prevalent in the afternoon (14:00–16:00 h), coinciding with daytimes of lower humidity and higher thermal amplitude. Adults that were submitted to lower temperatures (18°C) and short light periods (10 h) showed less propensity to flight. In contrast, at constant 27°C, the insects were more prone to flight, and this result was confirmed when individuals were submitted to increases in temperature, indicating that 27.14°C is the take-off temperature threshold of D. citri. Results show that temperature plays an important role in the flight activity of D. citri and suggest that control measures of the insect may be more effective in the morning and in temperatures below 27°C, when the probability to take off from a host is lower.