This study investigated the effects of different sucrose, ventilation, and paclobutrazol (PBZ) treatments on the growth of potato plantlets cultured in modified temporary immersion culture vessels. Temporary immersion culture vessels were modified to enable the plantlets to be cultured on semi-solid growth medium and provided with forced ventilation. The plantlet responses to two sucrose concentrations (15 g⋅L−1 and 30 g⋅L−1) in combination with two forced ventilation treatments (5 min/2 h and no ventilation) and three PBZ concentrations (0, 0.2, and 0.4 mg⋅L−1) were studied. Shoot growth was severely inhibited by PBZ in the growth media, whereas leaf formation was promoted by ventilation irrespective of the PBZ concentration. In nonventilated vessels, it is noteworthy that PBZ was able to increase the number of leaves formed in plantlets grown on medium supplemented with 30 g⋅L−1 sucrose, but not on medium with 15 g⋅L−1 sucrose. In growth media without PBZ, a high percentage of potato plantlets was able to produce secondary axillary shoots when provided with 30 g⋅L−1 sucrose. However, their ability to produce secondary shoots was reduced when PBZ was included in the growth medium, particularly those with 15 g⋅L−1 sucrose. Plantlets grown in ventilated culture vessels combined with 30 g⋅L−1 sucrose in the growth medium produced more than three times more shoots than the other treatments. Under ex vitro conditions, plantlets that had been grown in ventilated vessels had fewer leaf deaths, and the inclusion of PBZ in the growth media further reduced the number of dead leaves. Findings of this study showed that PBZ had a key role in the responses of potato plantlets to ventilation and sucrose treatments, as well as their tolerance to ex vitro conditions. The modified temporary immersion system can be used for the in vitro culture of potato plantlets on semi-solid medium and provide forced ventilation to improve their growth.