Transferring in vitro-cultured Eustoma seedlings to an ex vitro condition (acclimation) is a big challenge that may expose the seedlings to biotic and abiotic stresses, and affect the internal and external structure of the plants. In addition, in vitro-cultured seedlings of Eustoma are difficult to handle and phenotype and physiological traits such as survival and rosette rate may have altered in the acclimation stage. Therefore, the present study aims to examine the effects of blue, red, and white LED light on the growth and development ex vitro of in vitro-cultured seedlings of Eustoma. The results showed that blue LEDs resulted in greater plant height, internode length, and leaf number, increased upper and lower fresh biomass, and higher chlorophyll content compared with treatment by the other LED lights. Higher stomatal density on the abaxial leaf surface was also observed in the blue LED-treated plants, which also showed a higher survival rate and lower rosette rate. In contrast, the white LED-treated plants had the highest leaf width and internode diameter. Acclimation of the Eustoma plants ex vitro suggests that a combination of blue and white LEDs may be advantageous for better growth and development for large-scale production in a controlled environment.