Cultured meat technology is a novel and promising strategy for sustainable and effective meat production. Muscle stem cells are widely used as seed cell populations because of their ease of access and myogenic differentiation potential. However, muscle stem cells are difficult to continuously propagate ex vivo and are heavily dependent upon serum for survival and growth, which impedes their commercial use as a cultured meat source. Herein, we identified an effective four-cytokine combination to promote long-term proliferation of porcine muscle stem cells using a serial elimination screening approach, which was consisted of long chain human insulin growth factor-1, platelet derived growth factor BB, basic fibroblast growth factor, and epidermal growth factor. The expansion of muscle stem cells with the four-cytokine combination achieved a 6.31 × 107 -fold increase in cell number after 28 days of culture with retained cell myogenic differentiation potential, and most importantly, reduced the amount of fetal bovine serum required for cell culture by at least 5%. Furthermore, the four-cytokine combination exerted the pro-proliferative function by activating PI3K/Akt/mTOR and MEK/ERK signaling pathways. This approach provides for a new means by which to industrialize the production of cultured meat.