The Chinese hamster lung fibroblast cell line (CCI39), anchorage- and highly serum-dependent for growth, is tumorigenic in nude mice. Tumors arise after 4 to 8 weeks following the inoculation of 5 × 105 cells. We have shown that all the emerging tumoral clones (more than 20 analyzed) have lost the growth factor dependence of the parental cells (Pérez-Rodriguez et al., 1981 a). To mimic this selection which occurred in vivo, we selected in vitro growth-factor-independent variants. These variants, GFI304 and GFI461, can proliferate in a serum-free medium supplemented with transferrin alone. This character is stable since it is not lost after the GFI variants have been cultivated in non-selective medium (serum) for more than 20 generations. In spite of their “autonomous” growth and anchorage independence, GFI variants are poorly tumorigenic. The observation of nodule formation and subsequent regression in nude mice, immunosuppressed (irradiation or cyclophosphamide) or not, led us to the conclusion that at least two selections in vivo are required for the tumoral expression of CCI39 cells. One leads to a loss of growth factor requirement, the second towards a resistance to the immune surveillance mechanisms of the nude mice. The 4- to 8-week lag period of tumor formation may be accounted for by the spontaneous emergence in vivo of the two new characters necessary to bypass host growth restraints. This report supports the concept of stepwise progression and clonal evolution of preneoplastic cells in vivo and also indicates that tumorigenicity tests in nude mice should be interpreted with caution.