In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblast (NAFs), and normal prostate epithelial (PrE) cells; all three lines were immortalized by human telomerase reverse transcriptase (hTERT) alone and then were cryopreserved, thawed, and continuously passaged without any indication of a decrease in growth rate. Cell proliferation was measured under the influence of CAFs and NAFs for various prostate-derived epithelial cells. The effects of stromal cells on prostate cell proliferation was cell line-dependent. CAF cells promoted the growth of PrE, DU145, and A549, while NAF cells inhibited their growth. Meanwhile, CAF cells inhibited the growth of the viral transduced RWPE1 cell line, while NAF cells promoted the growth of the prostate epithelial cell LNCap. This study demonstrates that these hTERT-immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.