Simple SummaryCulturing of muscle precursor cells—satellite cells—is the basis for research on obtaining meat using in vitro techniques, which can become an alternative to traditional meat production. The available literature data do not give a clear answer as to the medium used at the proliferation stage of bovine satellite cells; therefore, there is a need to select the level of components of the growth medium. In our research, based on the fluorometric measurement of proliferating cell growth and qPCR analysis, we examined the effect of the three most common components: glucose, serum (bovine or horse), and bFGF—a mitogenic factor. The results show that the most critical component affecting the proliferation rate is the serum—the addition of bovine serum, followed by the addition of bFGF—at 10 ng/mL. In turn, the lower glucose content ensures the maintenance of cells at the early stage of myogenesis and, thus, the self-renewal of their population. In conclusion, a higher proliferation rate of bovine satellite cells is ensured under the conditions of bovine serum and a mitogenic factor at 10 ng/mL.AbstractIt is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells—being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor—bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p< 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.