Micropropagation is an asexual propagation tissue culture technique that is widely used to maintain genetically uniform and entirely female Cannabis clones. Stage II of micropropagation involves multiplication and cytokinins are regularly utilized to enhance multiplication rates and direct explants towards vegetative growth with hormonal stimulation. However, concerns have been raised about the negative impact of cytokinins on subsequent rooting and acclimation success during Stage IV. To address these challenges, a comprehensive two-part experiment was conducted using the cultivar ‘TJ’s CBD’. (1) Stage II in vitro experiments evaluated the effects of different cytokinin types and concentrations on explant growth, showing promising results for 6-Benzyladenine (BA) and 6-(γ,γ-Dimethylallylamino) Purine (2IP) and inhibitory effects for Thidiazuron (TDZ). (2) Stage IV ex vitro acclimation experiments revealed lingering inhibitory effects of TDZ on rooting success. Further research should establish the generalizability of the results among different Cannabis cultivars. Collectively, these findings contribute to optimizing cytokinin use during micropropagation, reducing costs, and enhancing productivity of Cannabis tissue culture laboratories.