Tumor initiating cells (TICs) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. We describe a novel approach to generate TICs by transduction of human primary breast cell preparations with exogenous expression of the OCT4 transcription factor. The OCT4 transduced breast colonies (OTBCs) underwent an immortalization process, as reflected by loss of p16 and enhanced telomerase expression. OTBCs exhibited cancer stem cell antigens, such as CD44high/CD24low/‐. These cells generated subcutaneous tumors in nude mice with colonization capabilities with 1–50 cells, thereby exhibiting TIC properties. Histological analysis of these tumors revealed poorly differentiated breast carcinomas. RNAi‐mediated knock‐down of OCT4 and downstream embryonic targets of OCT4 resulted in suppression of the self‐renewal ability. OCT4 over‐expression in these clones was molecularly associated with Epithelial‐to‐Mesenchymal Transition (EMT) gene signatures and strongly correlated with the claudin‐low intrinsic subtype of breast cancer. Our experimental approach provides a novel model system to dissect genes that could be used to effectively target breast cancer self‐renewal and tumor initiation.