The micropropagation of medicinal Cannabis (Cannabis sativa L.) offers an aseptic, scalable method for production and preservation of pharmaceutically elite Cannabis genotypes (strains) with specific chemical profiles. The present study investigated various factors influencing the establishment and progression of Cannabis through micropropagation. Four initial explant types (apical bud, second, fourth and sixth axillary nodes) and commercial genotypes (Amnesia, Glueberry Kush, Mokum’s Tulip and NZ Cheese) were used to investigate the establishment of Cannabis in vitro on 1⁄2 strength Murashige and Skoog (MS) medium. The in vitro progression of Cannabis on 1⁄2 MS medium across two rounds of plant tissue culture multiplication were investigated using secondary and tertiary explant material (shoot tips and axillary nodes) from responding apical bud explants across the four genotypes. The effect of plant growth regulator (PGR), meta-Topolin (m-T) at eight concentrations (0-5 μm/L) in MS medium supplemented with activated charcoal (0.5 g/L) was investigated using three genotypes (Amnesia, Glueberry Kush and Mokum’s Tulip), and the effect of activated charcoal was investigated using one genotype (Amnesia). The apical bud explant had the lowest rates of endogenous contamination (2%), highest rates of proliferation (97%) and the greatest provision of secondary plant material (3.84 average secondary explants). Genotype influenced the provision of secondary and tertiary explant material, proliferation response, canopy area, plant health and plant height. Plant health was affected by m-T concentration, with the best health achieved at 5 μm/L. Plants grown on PGR free MS medium had significantly higher shoot production, canopy area and plant height compared to media including m-T (at 2 and 5 μm/L). Activated charcoal supplementation was shown to negatively impact proliferation response, shoot production, canopy area and plant height. Findings from this study lay an important foundation for the development of a robust and replicable micropropagation system for medicinal Cannabis.