Transforming growth factor-beta (TGFbeta) superfamily ligands are produced by and act upon testicular cells to control testis morphogenesis and adult fertility. Ligand production changes during testis development and dysregulated signaling affects the number of cells comprising each lineage and their development, with several components of this diverse signaling pathway linked to male infertility. To test the hypothesis that TGFbeta superfamily signaling regulators are differentially expressed during mouse testis development, we surveyed expression of Hgs, Zfyve9, Smurf1 and Net25 by northern blot and in situ hybridization and SMURF2 and MAN1 by western blot and immunohistochemistry. Expression of these genes is highly regulated and differs between the first spermatogenic wave and adult spermatogenesis. Zfyve9 transcripts were first detected in Sertoli cells and spermatogonia at 5 days post partum (dpp) whereas Hgs mRNA was first detected in pachytene spermatocytes at 15 dpp. Smurf1 mRNA was broadly expressed at 0 and 5 dpp but restricted to spermatogonia and early spermatocytes at 15 dpp and spermatogonia, spermatocytes and round spermatids in adults. SMURF2 was limited to gonocyte nuclei at birth but was nuclear in all cells at 5 dpp. SMURF2 was absent from 15 dpp differentiating spermatogonia and early spermatocytes but readily detected in adult pachytene spermatocytes and round spermatids. MAN1 and Net25 also had different expression profiles, with MAN1 undetectable at 5 dpp. Differential synthesis of signaling modulators explains how Sertoli cells and spermatogenic cells, which all possess TGFbeta superfamily signaling machinery and reside within the same microenvironment, respond differently to the same ligand.