Our research focuses on the role of TCFL5, a bHLH transcription factor, in colon cancer cells. Deletion of TCFL5 significantly reduces tumor properties. Two major isoforms, TCFL5_E1/E8 and TCFL5_E2b/E8, have distinct functions. TCFL5_E1/E8 regulates anti-apoptotic genes like BCL2, while TCFL5_E2b/E8 controls pluripotency markers. We identified genes regulated by these isoforms and their roles in leukemia and lymphopoiesis. TCFL5_E2b/E8 is associated with severity in lymphoma and myeloma. Tcfl5 deficiency impairs germinal center formation and B cell differentiation.