In mammals, male or female development depends on testis or ovary formation, a process involving sex-specific transcription and signalling in the embryonic gonad. Disrupted testis and germline development is strongly associated with testis cancer in humans. In mice, Sry (Sex-determining region Y-chromosome) and Sox9 (Sry-box 9) drive Sertoli cell commitment and testis cord formation, committing the fetus to male development. SRY and SOX9 induce Sertoli cell expression of Fgf9 (Fibroblast growth factor 9), which is essential for testis development and is thought to promote male germ cell development and germ cell mitotic arrest. As FGF signals though the MAPK (Mitogen Activated Protein Kinase) pathway in other tissues, we explored whether FGF9 regulates male germline development through the MAPK pathway by inhibiting FGF9 or MEK1/2 signalling immediately after primary sex determination. Embryonic day (E)12.5 mouse testes from Oct4GFP transgenic mice were cultured with FGF9 or MEK1/2 inhibitors for 72 hours and the impacts on testis cord structure, Sertoli cell proliferation, germ cell mitotic arrest and testis and male germline developmental gene expression determined using whole-mount imaging, flow cytometry, immunofluorescent imaging and qRT-PCR. Testis cord structure was disrupted by either treatment, with Oct4GFP positive germ cells disorganised throughout the testis. While MEK1/2 signalling reduced Sertoli cell proliferation and disrupted germ cell mitotic arrest and expression of male germline markers, FGF9 inhibition only reduced Sertoli cell proliferation but did not inhibit germ cell mitotic arrest or male germline markers. Together, our data indicate essential roles for MEK1/2 signalling in testis development and male germline differentiation, but a more limited role for FGF9 signalling in somatic development of the testis. Moreover, our data strongly indicate that additional ligands promote male germline development through the MEK1/2 pathway and highlight a need for further understanding of the mechanisms underlying gonad development and testis cancer in humans.