Primary human squamous cell carcinomas (SCCas) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses. To determine if tumor-initiating cells (TICs) are present in SCCas, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCas, and demonstrated that a small subset of SCCa cells (∼1%) expressing Prominin-1 (CD133) in the outer layers of SCCas were highly enriched for TICs (∼1/400) compared with unsorted SCCa cells (TICs ∼1/10(6)). Xenografts of CD133+ SCCas recreated the original SCCa tumor histology and organizational hierarchy, whereas CD133- cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. We present a model of human SCCas in which tumor projections expand with outer leading edges that contain CD133+ TICs. Successful cancer treatment will likely require that the TICs identified in cancers be targeted therapeutically. The demonstration that TICs are present in SCCas and are enriched in a CD133- expressing subpopulation has not been, to our knowledge, previously reported.