There are several studies on the medicinal properties of dandelions (Taraxacum officinale), but few studies are aimed at understanding the in vitro germination process of this plant to improve its propagation. This research was focused on studying in vitro seed germination and development of seedlings under different carbon sources, glucose (GLU) or sucrose (SUC) and its concentrations (1.0–5.5%). Additionally, the effect of supplementation with plant growth regulators (PGRs) was studied, measuring the germination capacity, uncertainty and synchrony. Germination was promoted under low carbon source concentrations (≤2.3%), whereas higher concentrations (≥3.2%) had a detrimental effect on this process. GLU allowed the final germination percentages to be slightly better than SUC. Uniformity and synchrony values improved with the presence of PGRs. Results suggested that the best condition to assess T. officinale seed germination is in a medium containing GLU 2.3%, 0.5 mg/L 1-Naphthaleneacetic acid (NAA) and 0.5 mg/L 6-Benzylaminopurine acid (BAP). After germination, the best condition for optimal growth of T. officinale seedlings was 1.0% SUC supplemented with 0.225 mg/L of NAA and 3.0 mg/L of BAP for initial shoot development. The survival rate was 97% after greenhouse acclimatization. This new method of germination was implemented for the massive propagation of T. officinale for further medicinal studies.