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The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6 weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1 μM CdCl2 for 24 h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.