The details of the bifurcation of the lymphoid and myeloid lineages following commitment by multipotent progenitor cells (MPP) remain a topic of controversy. We report that the surface glycoprotein CD62L can be characterized as a novel marker of this and other stages of early hematopoietic differentiation. Cell isolation and transplant studies demonstrated CD62L(neg/low) long-term hematopoietic stem cells and CD62L(high) MPP within the traditionally defined c-kit(pos)Lin(neg/low)Sca-1(pos) stem/progenitor cell population. Within the MPP population, previously defined as c-kit(pos)Lin(neg/low)Sca-1(pos)-Thy-1.1(neg)Flt3(pos), Sca-1 and CD62L resolved four populations and segregated Sca-1(high)CD62L(neg/low) MPP from Sca-1(high)CD62L(high) leukocyte-biased progenitors. Using a novel transplantation method that allows tracking of erythroid and platelet engraftment as an alternative to the classical method of in vitro colony formation, we characterized Sca-1(high)CD62L(neg/low) cells as MPP, based on transient engraftment of these lineages. These data establish CD62L as a useful tool in the study of early hematopoiesis and emphasize the power of trilineage-engraftment studies in establishing the lineage potential of MPP subsets.