Currently, studies have analyzed the formation mechanism of primordial germ cell (PGC) at the transcriptional level, but few at the protein level, which made the mechanism study of PGC formation not systematic. Here, we screened differential expression proteins (DEPs) regulated PGC formation by label-free proteomics with a novel sampling strategy of embryonic stem cells and PGC. Analysis of DEPs showed that multiple key events were involved, such as the transition from glycolysis to oxidative phosphorylation, activation of autophagy, low DNA methylation ensured the normal formation of PGC, beyond that, protein ubiquitination also played an important role in PGC formation. Importantly, the progression of such events was attributed to the inconsistency between transcription and translation. Interestingly, MAPK, PPAR, Wnt, and JAK signaling pathways not only interact with each other but also interact with different events to participate in the formation of PGC, which formed the PGC regulatory network. According to the regulatory network, the efficiency of PGC formation in induction system can be significantly improved. In conclusion, our results indicate that chicken PGC formation is a complex process involving multiple events and signals, which provide technical support for the specific application in PGC research.