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Background Genetic and morphologic similarities between mouse embryonic stem cells (ESCs) and primordial germ cells (PGCs) make it difficult to distinguish differentiation of these two cell types in vitro. Using specific GC markers expressed in low level or even not expressed in ESCs- can help recognize differentiated cells in vitro. We attempted to differentiate the mouse ESCs into Gc-like cells spontaneously in monolayer and EB culture method. Materials and Methods In this experimental study, we attempted to differentiate ESCs, Oct4-GFP OG2, into GC-like cells (GCLCs) spontaneously in two different ways, including: i. Spontaneous differentiation of ESCs in monolayer culture as (SP) and ii. Spontaneous differentiation of ESCs using embryoid body (EB) culture method as (EB+SP). During culture, expression level of four GC specific genes (Fkbp6, Mov10l1, Riken and Tex13) and Mvh, Scp3, Stra8, Oct4 were evaluated. Results In both groups, Mov10l1 was down-regulated (P=0.3), while Tex13 and Riken were up-regulated (P=0.3 and P=0.04, respectively). Fkbp6 and Stra8 were decreased in EB+SP and they were increased in SP group, while no significant difference was determined between them (P=0.1, P=0.07). Additionally, in SP group, gene expression of Mvh and Scp3 were up-regulated and they had significant differences compared to EB+SP group (P=0.00 and P=0.01, respectively). Oct4 was down-regulated in the both groups. Flow-cytometry analysis showed that mean number of Mvh-positive cells in the SP group was significantly greater compared to ESCs, EB+SP and EB7 groups (P=0.00, P=0.01, and P=0.3, respectively). Conclusion These findings showed that ESCs were differentiated into GCLCs in both group. But spontaneous dif- ferentiation of ESCs into GCLCs in SP group (monolayer culture) compared to EB+SP (EB culture methods) has more ability to express GCs markers.