The fundamental question of how restriction of differentiation potential and activation of lineage-specific gene expression in progenitor cells are regulated is at the heart of research into the molecular mechanisms of biology. The basic helix-loop-helix (bHLH) transcription factor SCL/TAL-1 serves as a paradigm for a transcription factor with pivotal roles in haematopoietic stem cell (HSC) specification, maturation of haematopoietic lineages as well as T-cell leukaemogenesis. Interestingly, it has been described as both a transcriptional activator and a repressor. However, the molecular mechanisms conferring these opposing functions are currently unknown. As a first step towards characterising the role of SCL in haematopoiesis and leukaemogenesis, we performed a gain-of-function study using a transgenic mouse model over-expressing SCL. Although no leukaemia was observed, constitutive expression of SCL or a mutant lacking the DNA binding domain in HSCs and early progenitors caused a severe defect in T- and B-lymphopoiesis. This was likely due to an interaction with the important lymphoid regulator and SCL heterodimerisation partner E2A. Then, we set up a proteomics strategy to identify protein partners of SCL in haematopoiesis. We isolated SCL-containing protein complexes in erythroid cells and megakaryocytes using a novel purification strategy based on in vivo biotinylation of SCL. Mass spectrometry analysis led to the identification of several previously unknown candidate partners including ETO-2, a member of the ETO family of co-repressor proteins and SSDP2, a partner of LDB-1. Using several different approaches, we validated the SCL/ETO-2 interaction in cell lines as well as primary erythrocytes and megakaryocytes. We then identified Gfi-1 b, a critical regulator of erythropoiesis with repressive function and the co-repressor mSin3A as additional partners of SCL and ETO-2 in erythroid cells, but not megakaryocytes. Further characterisation in transactivation experiments and during in vitro differentiation of primary fetal liver cells led us to hypothesise that, in early erythropoiesis, ETO-2 might confer a repressive function to SCL. In conclusion, this study gives a first insight into how the composition of the multimeric protein complexes including SCL might modulate haematopoietic differentiation and cell fate decision.