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Gli1 is an important signaling molecular in Hedgehog signaling pathway. In our study, we explored the adjustment effect of Hedgehog-Gli1 signaling pathway on chicken male germ cells differentiation based on the transcriptome-wide analyses of chicken ESCs, primordial germ cells (PGCs) and spermatogonia stem cells (SSCs) that were associated with male germ cell differentiation. We screened out Hedgehog signaling pathway and identified 8 candidated differentially expressed genes (DEGs), Wnt3a, Wnt16, Wnt8a, HHIPL1, Gli1, BMP6, BMP7 and TTLL4. These DEGs expression change trend among blastoderm, genital ridge and testes, from which ESCs, PGCs and SSCs were isolated was the same as RNA-Seq data with quantitative RT-PCR evaluation. Based on retinoic acid (RA) induction of ESCs to SSCs in vitro, Gli1 overexpression has the ability to induce ESCs differentiation and SSCs-like cells formation and high expression of related reproductive genes, like Cvh, C-kit, Blamp1, Prmd14, Stra8, Dazl, integrin α6 and integrin β1 and so on in vitro. While RNAi knockdown of Gli1 can protect ESCs from differentiating into SSCs and correspondingly reduce the expression of the associated reproductive gene in vivo and vitro. Immunochemistry results showed that Gli1 overexpression could increase the expression of PGCs markers Cvh and C-kit and SSCs markers integrin α6 and integrin β1 in vivo, while Gli1 knockdown can have the opposite effect in vivo and in vitro. PAS stain and flow cytometry (FCM) evaluation results indicated the quantity of germ cells is decrease or increase with Gli1 knockdown or overexpression. Collectively, these results uncovered a novel function of Gli1 and demonstrated Hedgehog-Gli1 signaling pathway involved in chicken male germ cell differentiation, where it acts as a facilitator.