In Mexico the gladiolus (Gladiolus grandiflorus Andrews) asexual propagation is mainly carried out with corms and cormels produced by the farmers. This type of asexual reproduction is problematic when trying to obtain high quality corms because of the corm diseases spread more easily. Therefore, the aim of this study was to determine the most suitable environment for acclimatization of micropropagated Fusarium free plantlets. For in vitro propagation five different combinations of plant growth regulators were evaluated and for ex vitro acclimatization four environments were tested on a completely randomized experimental design. The highest rooted shoots percentage, number of roots, shoot length and 100% fungi-free plantlets were obtained when 0.2 mg l^-1 NAA was used. The best acclimatization environment was under 75% shading mesh which promoted the highest survival (96.8%), plant length (24 cm), corm biomass (1.2 g), total biomass (2.5 g plant^-1), and photosynthetic efficiency index (2.9). Additionally, the variable and maximum fluorescence ratio was nearly the optimal (0.83). This results could facilitate the large-scale production of Fusarium-free gladiolus plants without need be cultivated in a greenhouse conditions.