Due to the process of gonadal sex determination, the key supporting cell lineage differentiates into Sertoli cells in the testis or pre-granulosa cells in the ovary. Supporting cells are derived from a single sexually bipotential precursor lineage present in the early gonadal primordium. In mammals, it has been shown that the supporting cell progenitor line develops via ingression and EMT of cells from the coelomic epithelium. While the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell RNA sequencing in the embryonic chicken gonad during sexual differentiation. Combining lineage tracing with single cell transcriptomics, the data show that somatic supporting cells of the embryonic chicken gonad do not derive from the coelomic epithelium, in contrast to other vertebrates studied (mouse and turtle). Instead, the early somatic precursors cells of the gonads in both sexes derive from a resident DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. PAX2 was identified as a novel undifferentiated supporting cell marker in chicken, being downregulated at embryonic day (E) 6.0, consistent with the upregulation of DMRT1 expression in males and testicular differentiation. We were able to confirm PAX2 positive cells in quail, emu and zebra finch undifferentiated gonads, but not in mouse nor the bearded dragon (Pogona vitticeps). This suggests that the PAX2+ mesenchymal origin of supporting cells is conserved among birds, but not in reptiles or mammals. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.