Abstract Many agaves are economically important plants that have not been genetically improved, mainly due to their long life cycles. Micropropagation offers a means for increasing crop productivity of Agave species; however, an important limitation of this technique is the mortality rate of the in vitro cultured plants during transfer to soil. This is due to the morphological and physiological abnormalities produced by the physical and chemical conditions of in vitro culture. In order to understand these alterations, we investigated the development of the stomatal complex and of the leaf surface of Agave angustifolia shoots cultured under three different in vitro systems (semi-solid media [magenta boxes], permanent immersion [liquid in flasks with a cotton cover] and temporary immersion [BioMINT™ bioreactors]) and during the transition from in vitro to ex vitro conditions (greenhouse, shaded nursery and planted in the field). The apical, middle and basal regions of both the adaxial and abaxial leaf surfaces were analyzed by scanning electron microscopy (SEM) and the quantity and composition of leaf epicuticular waxes were analyzed by gas chromatography mass spectrometry (GC–MS). The leaf microstructure of the plantlets changed considerably through the sequential stages. While only immature stomata were observed in the in vitro phase, they developed to mature functional stomata in the various ex vitro phases. The results presented here indicate that temporary immersion system, may contribute to a pre-adaptation of plantlets, although they do not result in the formation of fully mature and functional stomata. The adaptation in the greenhouse and shaded nurseries is therefore convenient for the development of the plants.