Myc, a member of the "Myc Network" of bHLH-ZIP transcription factors, supervises proliferation, metabolism, and translation. It also engages in crosstalk with the related "Mlx Network" to co-regulate overlapping genes and functions. We investigated the consequences of stepwise conditional inactivation of Myc and Mlx in primary and SV40 T-antigen-immortalized murine embryonic fibroblasts (MEFs). -knockout ( KO) and × "double KO" (DKO)-but not KO-primary MEFs showed rapid growth arrest and displayed features of accelerated aging and senescence. However, DKO MEFs soon resumed proliferating, indicating that durable growth arrest requires an intact Mlx network. All three KO MEF groups deregulated multiple genes and functions pertaining to aging, senescence, and DNA damage recognition/repair. Immortalized KO MEFs proliferated in Myc's absence while demonstrating variable degrees of widespread genomic instability and sensitivity to genotoxic agents. Finally, compared to primary KO MEFs, DKO MEFs selectively downregulated numerous gene sets associated with the p53 and retinoblastoma (Rb) pathways and G /M arrest. Thus, the reversal of primary KO MEF growth arrest by either Mlx loss or SV40 T-antigen immortalization appears to involve inactivation of the p53 and/or Rb pathways.