The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNA-binding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant TAL1 protein expression in human hematopoietic CD34⁺ cells using a lentiviral vector. Differentiation capacities of the transduced cells were studied in a culture system that distinguishes early and late erythroid development. Results indicate that enforced TAL1 expression enhances long-term culture initiating cell (LTC-IC) potential and erythroid differentiation of human CD34⁺ cells as shown by increased βglobin and porphobilinogen deaminase (PBGD) gene expressions and erythroid colony-forming units (CFU-Es), erythroid burst-forming units (BFU-Es), and glycophorin A-positive (GPA⁺) cell productions. Enforced expression of a TAL1 protein deleted of its DNA-binding domain (named ΔbTAL1) mimicked most TAL1 effects except for the LTC-IC enhancement, the down-regulation of the CD34 surface marker, and the GPA⁺ cell production. These results provide the first functional indications of DNA-binding-dependent and -independent roles of TAL1 in human erythropoiesis. (Blood. 2004;103:3326-3335)