Although, many types of human leukaemia in now understood, the causes of human B-cell lymphocytic leukaemias are not clearly understood. Research showed that many genes that are required for B-cell development and maturation are mis-expressed in leukaemia, suggesting that alteration during B-cell development leads to transformation. Studies into the genetic causes of B-cell leukaemia on both humans and animal models are required to understand normal cellular development and improve treatments. Evi3 (ZFP521) is recently identified transcription factor with 30 zinc-finger domains which is over-expressed in recombinant inbred mouse strains with B-cell leukaemia. However, during normal B-cell development, Evi3 is expressed at all stages. In this study we aim to understand the role of Evi3 in B-cell development and leukaemia. In this study we have demonstrated that transcription factors HoxC13 and PU.1 are synergistically regulate Evi3 expression and they are expressed in immature and mature B-cells indicating that they may play a key role in regulating Evi3 expression. We have also confirmed that these transcription factors bind to their recognition sequences in Evi3 promoter. Furthermore, we have shown that knocking down Evi3 resulted in a reduction in cell viability and increased in the number of dead cells. Moreover, we have shown that genes regulating B-cell proliferation were down-regulated in cells with Evi3 knockdown. Pax5 is a transcription factor that is essential for the later development of B-cell. Furthermore, Pax5 is a potential Evi3 target gene and its expression was shown to be upregulated in tumour cells with viral insertion at Evi3 locus. Additionally, we have demonstrated that co-transfection of Evi3 and Ebf1 may upregulate Pax5 expression. Given together, these new experiments will provide a key insight on the role of Evi3 in B-cell development and B-cell proliferation. Hence, Evi3 could be a new possible target for treatment in B-cell leukaemia.