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Germ cell production from stem cells allows for studying the mechanisms involved in gamete development with the aim of helping infertile couples with the generation of healthy gametes. In this context, improving the protocols for in-vitro germ cell induction from stem cells is very important. Recently, SB4 small molecule has been introduced as a potent agonist for bone morphogenic protein 4 (BMP4). Herein, we investigated whether BMP4, is replaceable by SB4 for having affordable protocol for in vitro germ cell differentiation. We demonstrated that SB4 can induce Blimp1 (as the first gene induced germ line differentiation) expression significantly but at a lower level compared to BMP4. However, Tfap2c (a putative downstream target of Blimp1 during germ cell differentiation) expression level in SB4-induced aggregates was significantly higher than in BMP4-induced aggregates. Moreover, co-presence of both BMP4 and SB4 could increase the expression level of Prdm14, Nnose3 and Stella (Dppa3), and thereby improve establishment of the germ cell fate during in-vitro differentiation of embryonic stem cells. In summary, our data suggest that SB4 could improve germ line gene expression pattern induced by BMP4 during embryonic stem cells in-vitro differentiation., (Copyright © 2021. Published by Elsevier B.V.)