<p>Blueberries (<i>Vaccinium</i>), belonging to the <i>Ericaceae</i> family, are propagated using strategies such as micropropagation to optimize clonal production. From this perspective, this study aimed to improve the acclimatization process of blueberry plants by evaluating the effect of different flask sealing methods during in vitro cultivation. Three cultivars—Bluegem, Climax, and Woodard—were used in the experiment. Explants were induced to root in a medium composed of water, 5.5&#xa0;g L⁻¹ agar, and 20&#xa0;mg L⁻¹ indole-3-butyric acid (IBA), then transferred to flasks containing Woody Plant Medium (WPM) supplemented with activated charcoal. The cultures were maintained in a growth chamber for 30 days under three sealing conditions: standard lids, plastic film, and lids with filters. Following this period, explants were transplanted to polyethylene trays containing a commercial substrate and acclimatized in a greenhouse equipped with an intermittent microspray system for 60 days. The experiment included morphometric, histological, survival, and photosynthetic pigment analyses. Results varied among cultivars. No significant differences in morphometric parameters were observed among the three cultivars during the in vitro proliferation stage. However, differences emerged in survival rates and in morphometric and pigment-related parameters at the end of acclimatization. The use of filter lids improved the micropropagation and acclimatization efficiency of the Climax cultivar. Additionally, filter-sealed flasks enhanced survival rates in Bluegem, whereas no notable improvements were observed for the Woodard cultivar. These findings highlight the importance of optimizing flask sealing systems in tissue culture to develop more efficient acclimatization protocols, thereby increasing the quantity and quality of plantlets obtained.</p>