The role of epidermal basal stem cells in dysplasia is a matter of great interest in the human papillomavirus (HPV)-driven cancers. To assess the relationship between “stemness” and HPV-mediated transformation, we made use of 3-D suspension culture and fluorescence activated cell sorting (FACS) to purify stem/progenitor-like cells from primary normal human keratinocyte (NHKc) cultures. We found that NHKc cells derived from multicellular keratinocyte spheroids were enriched for a basal subpopulation of epidermal stem-like cells, that could be maintained for prolonged time in culture and used to conduct transfection experiments with full-length HPV16 DNA. Thus, by using these stem cell enrichment methods, we set out to investigate in detail the effects of increased and decreased basal stem cell density on keratinocytes’ immortalization and transformation efficiencies. We hypothesized that stem cell properties of NHKc cultures established from neonatal genital skin would positively influence susceptibility to transformation by HPV16 DNA. Our findings reveal that epidermal stem cells (EpSCs) are more effectively immortalized and transformed by oncogenic HPV16 DNA, while terminally differentiated keratinocyte populations fail to successfully immortalize in culture. Tissue stem cell density may prove useful in predicting individual susceptibility to HPV16-mediated transformation in persons with persistent HPV infections, improving on current triage and follow-up measures.