In vitro culture systems are needed to mimic in vivo epithelial cell environments for identifying cell signaling, gene expressions, and molecular mechanisms. One such system is matrix-coated transwell cultures. However, no data exist on culturing Sertoli cells in this manner with respect to testosterone-regulated gene expression. Because the TM4 mouse Sertoli-like cell line expresses androgen receptor, our objective was to determine if testosterone treatment added to the bottom chamber of a matrix-coated transwell system induces some gene expressions found in Sertoli cells in vivo. After serum starvation, transwell-cultured TM4 cells were treated with testosterone or left untreated for 24 h. Microarray analyses initially identified differentially expressed genes either induced or repressed by testosterone treatment. By Northern blot analyses, Pem mRNA, a frequently used marker of Sertoli cell testosterone responsiveness, was induced. Proteins of the transcripts induced by testosterone in the in vitro system were immunolocalized to Sertoli cells in testosterone-dependent stages of spermatogenesis in mouse testes. By immunohistochemistry analyses of sectioned mouse testes, gene expression induced by testosterone in transwell-cultured TM4 cells, profilin as well as secreted protein acidic and rich in cysteines (SPARC) are localized to Sertoli cells in testosterone-dependent stages of spermatogenesis. Findings include localizations of SPARC and profilin, as well as an apparent germ cell communication required for translation of Pem mRNA in Sertoli cells. Taken together, results of these studies suggest that this TM4 transwell-culture system could be used to study these testosterone-regulated Sertoli gene expressions in vitro.