Autophagy plays contrasting roles depending on the stage of cellular transformation. However, although advanced tumor cell models are abundant, cell lines at the initiation stage of transformation are very limited. Therefore, the development of initiated cell lines-cells that have acquired early genetic alterations but not yet completed the multistep transformation process -is crucial for the development of anticancer drugs targeting autophagy. In this study, we successfully established a new initiated cell line (Foci #3) from foci formed in the in vitro two-stage cell transformation assay with NIH3T3 fibroblast cells. Foci #3 cells retained typical features of epithelial morphology, similar to its parental untransformed NIH3T3 cells. However, unlike NIH3T3 cells, where many dead cells were found during the long-term culture of 40 days, few dead cells were observed in Foci #3 cells. Particularly, the sensitivity of Foci #3 cells to the autophagy inhibitor CQ was higher than that of NIH3T3 cells and similar to that of Bhas 42 cells, the most commonly used initiated cell line. Moreover, Foci #3 cells maintained a higher level of autophagic flux than NIH3T3 cells throughout the extended culture period, indicating acquisition of the characteristic of dependence on autophagy for survival, which is typical of transformed cells. Importantly, qPCR analysis of epithelial-mesenchymal transition gene expression revealed that the Foci #3 cell line exhibited characteristics of both non-tumorigenic and tumorigenic states. Whole-genome sequencing analysis revealed that among the 17 genes with exon mutations in the Foci #3 cells, four were tumor suppressors and eight were involved in oncogenesis. Additionally, the Foci #3 cell line exhibited the loss of copy number variations in several tumor suppressors. Together, our results suggest that the newly developed Foci #3 cell line may be an efficient tool for elucidating the role of autophagy in the early stages of transformation.