The signalling milieu in the developing testis is complex, involving many TGFβ superfamily ligands. Elucidating how fetal male germ cells (gonocytes) differentiate in response to the somatic environment will aid our understanding of how male infertility and testicular germ cell tumours arise. This study examined the potential influence of activin A and other TGFβ superfamily ligands on germ cell differentiation during the important developmental window after sex determination. Whole embryonic (E)13.5 mouse testes expressing a germ cell-specific Oct4-GFP transgene were cultured for 48 hrs in media containing 10 µM SB431542, an activin/Nodal/TGFβ inhibitor, or in 5 ng/mL activin A, with appropriate vehicle controls. Transcripts were measured by qRT-PCR (n=5), and germ and Sertoli cell proliferation measured following EdU incorporation, antibody staining and flow cytometry (n=3). To assess direct actions on germ cells, E13.5 gonocytes isolated by FACS were cultured for 24 hrs in these conditions and collected for transcript analysis (n=5-6). SB431542-treated testes had obviously altered cord structure, and although Sox9 transcript levels were higher in this group, no change in Sertoli cell proliferation was measured. A significantly higher number of germ cells escaped mitotic arrest (2.7%) compared to vehicle control (<1%). Germ cell differentiation-associated genes Nanos2, Dnmt3a, Dnmt3l and Piwil4 were significantly lower, and the early germ cell marker Kit was significantly higher. The only change identified in activin A-treated testes was a 10% reduction of Sox9. In cultures of isolated gonocytes, SB431542 treatment resulted in significantly higher Kit and lower Nanos2 levels, while activin A exposure resulted in the reciprocal outcomes of significantly lower Kit and significantly higher Nanos2. These data suggest that activin/Nodal/TGFβ inhibition delays germ cell differentiation in whole gonads and gonocyte cultures, and that gonocytes can respond directly to changes in activin A and TGFβ superfamily signalling, with activin A promoting a more differentiated phenotype.