Stage-specific differentiation markers were used to evaluate the cellular composition and the origin of nonimmortalized (PrEC) and immortalized (PZ-HPV7, CA-HPV10, RWPE-1, and 957E/hTERT) human prostate cell lines. These studies documented that immortalized and nonimmortalized prostate epithelial cells established and maintained in low (i.e., <300 μmol/L) Ca²⁺ serum-free defined (SFD) medium were all derived from normal nonmalignant prostate tissues and contain CD133⁺/ABCG2⁺/α₂β₁^Hi/p63⁻/PSCA⁻/AR⁻/PSA⁻ prostate stem cells. In these cultures, prostate stem cells are able to self-renew and generate two distinct cell lineages: the minor proliferatively quiescent neuroendocrine lineage and the major transit-amplifying cell lineage. Subsequently, CD133⁻/ABCG2⁻/α₂β₁^Hi/p63⁺/PSCA⁻/AR⁻/PSA⁻ transit-amplifying cells proliferate frequently and eventually mature into proliferatively quiescent CD133⁻/ABCG2⁻/α₂β₁^Lo/p63⁻/PSCA⁺/AR⁻/PSA⁻ intermediate cells. Such proliferatively quiescent intermediate cells, however, do not complete their full maturation into CD133⁻/ABCG2⁻/α₂β₁^Lo/p63⁻/PSCA⁻/AR⁺/PSA⁺ luminal-secretory cells in low Ca²⁺ SFD medium. Addition of universal type I IFN and synthetic androgen (R1881) to culture medium resulted in up-regulation of androgen receptor protein expression. However, it failed to induce full differentiation of intermediate cells into AR⁺/PSA⁺ luminal-secretory cells. Our results indicate that such inability of prostate epithelial cells to complete their differentiation is due to continuous expression of Notch-1 receptor and its downstream effector, Hey-1 protein, which actively suppresses differentiation via its ability to transcriptionally repress a series of genes, including the GATA family of transcription factors. (Cancer Res 2006; 66(17): 8598-607)