In multiunit cord blood transplantation, hematopoietic stem cells from each unrelated cord blood (UCB) unit competitively reconstitute the hematopoietic system in a recipient. To evaluate the fate of the progeny of each UCB unit and to determine the effects of graft-versus-graft reaction, we established a novel competitive repopulation assay using NOD/SCID/gammac(null) mice in which human T lymphocytes develop from CD34+ cells. CD34+ cells from each UCB unit were labeled with recombinant lentivirus vectors carrying genes encoding either enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP). Hematopoietic chimerism composed of both EGFP+ and EYFP+ cells was stably maintained up to 6 months after transplantation with purified CD34+ cells; the ratio of EGFP+ to EYFP+ cells in peripheral blood and bone marrow posttransplantation was equivalent to the ratio of these cells at transplantation. However, when mononuclear cells from two UCB units were cotransplanted with CD34+ cells, engraftment was highly competitive, with cells from only one or the other of the two UCB units surviving. Further subfractionations of mononuclear cells indicate that the skewed chimerism that is often observed in clinical multiunit cord blood transplantation may be mediated by the cooperation of both CD4+ and CD8+ T cells. The assay established here will be a useful tool for analyzing hematopoietic reconstitution in clinical multiunit cord blood transplantation.