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Summary Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease., Graphical Abstract, Highlights • Discrete, proximal stem cell niche domains in juxta-urethral ducts • Basal stem cells generate large-scale, directed, and cohesive cell migration streams • Bipotent basal progenitors maintain the luminal compartment • DLK1 marks stem cells in situ that recapitulate gland histology in 3D culture, Moad et al. find that multipotent prostate basal stem cells, marked by delta homolog 1 (DLK1), reside in proximal ducts and generate directed large-scale epithelial flows traversing the entire length of the branching gland network. This work describes mechanisms underlying 3D epithelial homeostasis in a complex branching tissue.