Bidirectional signaling between the urogenital sinus epithelium and mesenchyme is an essential element of prostate development that regulates ductal morphogenesis, growth, and differentiation. Comparable interactions between the epithelium and stroma in the adult prostate appear to regulate normal growth homeostasis. Alterations in the stromal-epithelial dialogue that recapitulate features of the mesenchymal-epithelial interactions of development may play a critical role in the development of benign prostatic hyperplasia and in the progression of prostate cancer. For this reason, the mesenchymal-epithelial interactions of development are of considerable interest. In this review, we provide an overview of the mesenchymal contribution to rodent prostate development with an emphasis on the stage just before ductal budding (embryonic day 16; E16) and describe the isolation, characterization and utility of a newly established E16 urogenital sinus mesenchymal cell line.