Our study aimed to explore the regulatory mechanism of the carbohydrate metabolism signaling pathways and related genes during the differentiation of chicken embryonic stem cells to male germ cells, providing the basis for improving the efficiency of the in vitro induction system. Cell sorting was used to obtain highly purified embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each cell type. The transcriptions of ESCs, PGCs, and SSCs were sequenced by DNA microarray and mRNA sequencing (RNA-seq). The results were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The key pathways and genes of carbohydrate metabolism were screened during the differentiation process of chicken male germ cell. We concluded that 419 differentially expressed genes enriched to 26 carbohydrate metabolism pathways during the differentiation process of ESCs to SSCs, all of the chondroitin sulfate (CS) signaling pathway was significant. We screened the key genes CHSY3, B3GAT1, CHPF, and B4GALT7 which was significantly expressed in CS pathway. Quantitative RT-PCR showed that the expression trend of these genes is consistent with DNA Microarray and RNA-seq results. Our study supports the opinion that CS pathway is significantly different during the differentiation of chicken male germ cell (P < 0.05) and that CHSY3, B3GAT1, CHPF, and B4GALT7 are key genes.