Acclimatization of in vitro plantlets is one of the key steps in successful tissue culture propagation. Gaseous atmosphere during in vitro culture can influence the rate of ex vitro acclimation of the plantlets produced. In the current study, effects of elevated CO2 concentration on the leaf water loss dynamic responses of in vitro–produced walnut leaves during ex vitro desiccation were investigated. Elevated CO2 concentration in the headspace of culture vessels caused a considerable decrease in stomatal aperture. Although the traits related to stomatal size were not influenced by CO2 elevation, the number of small stomata was increased, and the number of large stomata was decreased at elevated CO2 concentration. Higher CO2 concentration resulted in a lower transpiration rate and a higher relative water content (RWC) during ex vitro desiccation. This improvement was due to decreased stomatal aperture during the first phase of water loss. Osmotic potential (ψs) was decreased under an elevated CO2 concentration, but no influence was observed on the concentration of compatible solutes. In conclusion, increasing the CO2 concentration of culture vessel headspace can be an efficient tool for improving acclimation of in vitro–grown walnuts without negative effects on plantlet growth.