CD34⁺Thy-1⁺Lin⁻ cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34⁺Thy-1⁺Lin⁻ cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34⁺Thy-1⁺Lin⁻ cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34^hiPKH^lo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34⁺Thy-1⁺Lin⁻ cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34^hi PKH^hi cells retained a high frequency of CAFC, whereas the small population of CD34^hiPKH^lo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34⁺Thy-1⁺Lin⁻ cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.