DNA hypermethylation in gene promoters is an epigenetic mechanism regulating gene expression in cellular immortalization, an important step in carcinogenesis. Previously, we studied the genes dysregulated during immortalization using spontaneously immortalized fibroblasts from patients with Li-Fraumeni syndrome (LFS), who carry a germline mutation in the tumor suppressor gene p53. We found that multiple interferon (IFN) signaling pathway genes were regulated by epigenetic silencing. In this study we focused on a key regulator of that pathway, the signal transducer and transcription activator 1 (Stat1) gene. Although Stat1 is downregulated after cellular immortalization and upregulated in immortal MDAH041 cells after 5-aza-2'-deoxycytidine (5-aza-dC) treatment, we detected no methylation of the Stat1 promoter region in these cells before or after immortalization. To analyze the function of Stat1 in immortalization, we expressed Stat1 in immortal MDAH041 cells by stable infection, expecting to induce IFN-regulated genes or cellular senescence or both. However, the overexpression of Stat1 alone was not sufficient to repress the proliferation rate of immortal MDAH041 cells or induce senescence in immortal MDAH041 cells. We concluded that factor(s) additional to Stat1 (whether IFN dependent or not) are required for the immortalization of LFS fibroblasts.