To address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34(+)CD38(lo) progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34(+) cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are "primed" for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.