Gonadal sex differentiation – testis versus ovary formation - is a fundamental process required for reproduction and evolution. Reflecting this importance, the embryonic gonads of vertebrate species comprise the same major cell types; germ cells, supporting cells and interstitial steroidogenic cells. Remarkably, the genetic triggers for gonadal sex determination varies across species (the SRY gene in mammals, DMRT1 in birds and some turtles, temperature in reptiles, AMH and various other genes in fishes). Despite this variation, the cell biology of gonadal development was long thought to be largely conserved. Recent single cell RNA-seq studies have shed light on cell lineage specification in the mouse, through the analysis of SF1 (Nr5a1) sorted somatic cells. Here, we present a more comprehensive analysis of gonadal sex differentiation, using the chicken embryo as a model and considering the entire gonad. We sampled over 40,000 cells across several developmental stages, prior, during and after the onset of gonadal sex differentiation. The data provide several new insights into cell lineage specification during vertebrate gonadogenesis. Firstly, the data reveal a greater complexity of gonadal cell types than previously thought. Secondly, unlike in mouse, the Sertoli cell lineage of the embryonic chicken gonad does not derive from the coelomic epithelium. Thirdly, we identify the cell lineage expressing the key avian sex determinant, DMRT1, and the gene regulatory network that it engages. DMRT1 governs avian sex determination, but its transcriptional targets are unknown. The data identify those transcripts co-expressed with DMRT1 in the developing gonad, providing and avenue for further analysis. These results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification may also vary.