Abstract Ectopic expression of transcription factors (TFs) can drive differentiation of embryonic stem cells, trans- differentiation between different somatic cell lineages, and reprogramming of somatic cells into induced pluripotent stem cells. This observation underscores the central role transcriptional factors (TFs) play in gene regulatory networks to dictate cellular phenotypes. The endogenous activities of TFs can be dramatically altered through mechanisms such as alternative splicing of TF messenger RNAs, in which multiple, functionally distinct protein isoforms can be produced from the same gene. Distinct TF isoforms can exhibit differential abilities to bind DNA targets, cofactors, or chromatin-associated proteins, resulting in complementary or opposing functions. For example, FOXP1 produces two isoforms that differ in their DNA-binding specificities: one activates self-renewal pathways, while the other activates differentiation pathways. On a global scale, such isoform-driven functional rewiring is emerging as a major regulatory strategy that controls differentiation and development. However, reliably detecting and discovering biologically relevant TF isoforms is challenging, particularly at the proteome-scale. To characterize TF isoforms that play a role in stem cell differentiation, my lab will apply a powerful combination of analytical and systems biology approaches to identify and experimentally characterize the functional consequences of TF isoforms. This project will involve several interlinked areas, including goals to: 1) discover and quantify full-length TF isoforms associated with differentiated cell states, 2) find TF isoform “drivers” of differentiation, and 3) uncover the mechanism(s) by which TF isoforms exhibit different functions. Our approach will use proteogenomic analysis that integrates long-read sequencing and MS-based proteomics data, high- throughput functional screening of large-scale isoform libraries, and characterization of molecular determinants (e.g., protein binding, DNA binding) and activities (gene activation or repression) that underlie differential TF isoform function. Collectively, the resulting data will enable discoveries of new general mechanisms of cellular differentiation and their drivers. This research program will provide a fundamental understanding of how TF isoforms function in regulatory networks and will contribute to the field by expanding the toolkit of TFs used in stem cell engineering applications. More broadly, this framework offers a new paradigm to enable isoform-resolved biological investigations, which could accelerate discovery of key isoform regulators in physiological and pathological states.