We report here that in chronic lymphocytic leukemia (CLL), the propensity to generate clonal B cells has been acquired already at the hematopoietic stem cell (HSC) stage. HSCs purified from patients with CLL displayed lymphoid-lineage gene priming and produced a high number of polyclonal B cell progenitors. Strikingly, their maturation into B cells was restricted always to mono- or oligo-clones with CLL-like phenotype in xenogeneic recipients. These B cell clones were independent of the original CLL clones because they had their own immunoglobulin VDJ genes. Furthermore, they used preferentially VH genes frequently used in human CLL, presumably reflecting the role of B cell receptor signaling in clonal selection. These data suggest that HSCs can be involved in leukemogenesis even in mature lymphoid tumors. ► HSCs in CLL have cell-intrinsic propensity to generate clonal CLL-like B cells ► Abnormal karyotypes are not required for appearance of such B cell clones ► The majority of single HSCs in patients with CLL display lymphoid-lineage priming ► HSCs can be involved in oncogenesis even in mature lymphoid tumors