Fas, an important death receptor-mediated signaling pathway, has been shown to be down-regulated during human colon tumorigenesis; however, how alterations in Fas expression influence the metastatic process remains unresolved. In mouse models, loss of Fas function was found to be both necessary and sufficient for tumor progression. In this study, we investigated the link between functional Fas status and malignant phenotype using a matched pair of naturally occurring primary (Fas-sensitive) and metastatic (Fas-resistant) human colon carcinoma cell lines in both in vitro and in vivo (xenograft) settings. Metastatic sublines were produced in vitro from the primary tumor cell line by functional elimination of Fas-responsive cells. Conversely, sublines derived from the primary tumor in vivo at distal metastatic sites were Fas-resistant. In contrast, simply disrupting the Fas pathway by molecular-based strategies in the Fas-sensitive primary tumor failed to achieve the same metastatic outcome. Interestingly, both in vitro- and in vivo-produced sublines resembled the naturally occurring metastatic population, based on functional and morphologic studies and genome-scale gene expression profiling. Overall, using this human colon carcinoma model, we: 1) showed that loss of Fas function was linked to, but alone was insufficient for, acquisition of a detectable metastatic phenotype; 2) demonstrated that metastatic subpopulations pre-existed within the heterogeneous primary tumor, and that anti-Fas interactions served as a selective pressure for their outgrowth; and 3) identified a large set of differentially expressed genes distinguishing the primary from metastatic malignant phenotypes. Thus, Fas-based interactions may represent a novel mechanism for the biologic or immunologic selection of certain types of Fas-resistant neoplastic clones with enhanced metastatic ability.