Simple SummaryThe present study examined the potential benefits of male specific-pathogen-free (SPF) White Leghorn embryos in cellular agriculture for sustainable and ethical poultry meat production—addressing traditional farming challenges, including disease outbreaks ofSalmonellaandAvianinfluenza. We generated myogenic precursor cells (MPCs) from the thigh muscles of embryos from SPF White Leghorns that tested negative forSalmonella. When male and female embryonic MPCs were cultured in vitro, it was confirmed that genetic sex did not affect the expression of factors related to myogenic regulation factors. Therefore, MPCs from male SPF-laying chicken embryos are promising for developing clean animal-cell-derived protein sources via resource recycling.AbstractThis study examined the potential benefits of male specific-pathogen-free (SPF) White Leghorn embryos in cellular agriculture for sustainable and ethical poultry meat production—addressing traditional farming challenges, including disease outbreaks ofSalmonellaand Avian influenza. We isolated myogenic precursor cells (MPCs) from the thigh muscles (Musculus femoris) of 12.5-day-old embryos from 10 SPF White Leghorns that tested negative forSalmonella. We randomly selected MPCs from three males and three females, isolated them using a modified pre-plating (pp) method, and compared their in vitro development. After 1 h (pp1) and 2 h (pp2) of incubation, they were transferred to a new dish to remove fast-adhering cells and cultured (pp3). Isolated MPCs had a 69% positive reaction to Pax7. During proliferation, no differences were observed inPAX7, MYF5, orMYODexpression between the male and female MPCs. However, after five days of differentiation, the expression of late myogenic factors—MYOGandMYF6—significantly increased in all MPCs. Notably,MYOGexpression was 1.9 times higher in female than in male MPCs. This impactedMYMK’s expression pattern. Despite this, the myotube fusion index did not differ between the sexes. Muscle cells from male SPF-laying chicken embryos are promising for developing clean animal-cell-derived protein sources via resource recycling.