During differentiation and development, cell fate and identity are established by waves of genetic reprogramming. Although the mechanisms are largely unknown, during these events, dynamic chromatin reorganization is likely to ensure that multiple genes involved in the same cellular functions are coregulated, depending on the nuclear environment. In this study, using high-content screening of embryonic fibroblasts from a beta-actin knockout (KO) mouse, we found major chromatin rearrangements and changes in histone modifications, such as methylated histone (H)3-lysine-(K)9. Genome-wide H3K9 trimethylation-(Me)3 landscape changes correlate with gene up-and down-regulation in beta-actin KO cells. Mechanistically, we found loss of chromatin association by the Brahma-related gene (Brg)/Brahma-associated factor (BAF) chromatin remodeling complex subunit Brg1 in the absence of beta-actin. This actin-dependent chromatin reorganization was concomitant with the up-regulation of sets of genes involved in angiogenesis, cytoskeletal organization, andmyofibroblast features in beta-actin KO cells. Some of these genes and phenotypes were gained in a beta-actin dose-dependent manner. Moreover, reintroducing a nuclear localization signal-containing beta-actin in the knockout cells affected nuclear features and gene expression. Our results suggest that, by affecting the genome-wide organization of heterochromatin through the chromatin-binding activity of the BAF complex, beta-actin plays an essential role in the determination of gene expression programs and cellular identity.