Mesonephric cell migration and seminiferous cord formation are critical processes in embryonic testis development at the time of male sex determination. Extracellular growth factors shown to influence seminiferous cord formation such as neurotropin-3 utilize in part the phosphotidylinositol 3-kinase (PI3K) signal transduction pathway. The current study investigates the hypothesis that the PI3K pathway is critical in seminiferous cord formation and testis development. The role of the PI3K signaling pathway in testicular cord formation was examined using an Embryonic Day 13 organ culture system and a PI3K-specific inhibitor LY294002. The actions of a mitogen-activated protein (MAP) kinase-specific inhibitor PD98059 was also examined. The PI3K inhibitor blocked cord formation or reduced the number of cords in a concentration-dependent manner. The actions of LY294002 were found to have a developmental stage specificity in that cord inhibition was observed in organs from embryos with 16–17 tail somites, while organs from embryos with 19 or more tail somites had no block in cord formation and only a small reduction in cord number. In contrast, the MAP kinase inhibitor PD98059 did not block cord formation and only caused a slight reduction in cord number. Neither PI3K or MAP kinase inhibitor altered apoptotic cell number, suggesting apoptosis was not the reason for the inhibition of cord formation. Embryonic testis cell migration assays showed that the PI3K inhibitor LY294002 blocked mesonephros cell migration into the testis, while the MAP kinase inhibitor had no effect. Observations suggest the interference of cell migration is the cause for the inhibition of cord formation. Western blot analysis confirmed that LY294002 and PD98509 inhibited phosphorylation of Akt and ERK1/ERK2, respectively. Combined observations demonstrate that the PI3K signaling pathway is involved in embryonic testis cord formation and mesonephros cell migration.