ObjectiveLittle is still known about the origin of somatic progenitor cells of human fetal gonads, their molecular identity and the transcriptional programs controlling their differentiation towards multiple fates. In this study, we performed single-cell RNA-sequencing to characterize cell lineage progression during gonad differentiation and to build the first atlas of human fetal gonads at single cell resolution.Material and methodologiesHuman fetal gonads (n = 15 females,n = 15 males) from 6 to 12 postconceptional weeks (PCW) were freshly dissociated and single cells were captured using the Chromium system (10X Genomics). Resulting sequencing libraries were sequenced and data analysis was performed in several steps including quality controls, normalization, dimensionality reduction, clustering and pseudotemporal cell trajectory inference.ResultsWe present a single cell expression atlas of human gonad development comprising 123,869 cells. A close cell composition was identified in female and male gonads at 6 PCW, followed by rapid lineage- and sex-progression during gonad differentiation. Furthermore, eight major trajectories were revealed during differentiation of supporting and interstitial lineages.DiscussionThis single-cell atlas confirms the complexity of cell lineages which differentiation is strikingly asynchronous in female and male developing gonads. We identified common gonadal somatic progenitor cells at 6 PCW that progressively commit towards either the steroidogenic or the supporting fate, in both female and in male gonads. This atlas will provide a valuable resource to deepen our knowledge on the molecular and cellular biology of normal human gonad development and to improve the identification of gene variants likely implicated in DSD patient phenotypes.