Ikaros is a transcription factor that acts both as an activator and as an inhibitor of gene expression in several hematopoietic lineages. Ikaros functions in hematopoiesis have mostly been studied in mice, and are notably crucial for lymphopoiesis. Deregulation of Ikaros expression was evidenced in several leukemia subtypes, including pre-B-ALL. Here, we studied the role of Ikaros in human B lymphoid differentiation through xeno-transplantation of genetically modified cord blood (CB) human hematopoietic progenitor cells (HPC) in NOD/SCID mice. We used lentiviral vectors to force expression of Ikaros 6 (Ik6), a known dominant negative (DN) protein that interferes with normal Ikaros and structurally related proteins in HPC and their progeny. Two types of vectors were used: a vector containing the EF1alpha promoter which produces strong gene expression in all hematopoietic lineages, and a recently validated B-specific vector containing an enhanced CD19 derived promoter that strongly favors expression in the B-cell lineage. Ik6 transduction of CB CD34(+) cells with these vectors produced distinct consequences in the B-cell differentiation profiles of xenografted human cells. While the ubiquitous vector favored a specific block at the early pro-B/pre-B stage of differentiation, with an increase in Lambda Like transcript expression in the bone marrow (BM), B-specific Ik6 expression provoked a global decrease in the CD19(+) cell population in both BM and spleen, associated with a decrease in IgM+ immature B-cells in the spleen. We conclude that Ikaros proteins are active throughout human B-cell differentiation, before and after CD19 appearance.