Dendritic cells (DCs) are powerful antigen-presenting cells. Because DCs are rare cells, methods to produce them in vitro are valuable ways to study their biologic properties and to generate cells for immunotherapy. This study defines the antigen-presenting properties of DCs generated in vitro from CD34+cells of patients with breast cancer. The combination of cytokines flt3 ligand + c-kit ligand + granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) + tumor necrosis factor-&agr; (TNF-&agr;) was used to maximize the output of mature DCs in the culture of CD34+cells while minimizing the production of monocytes. Cells grew and differentiated into DCs as measured by a time-dependent upregulation of cell surface antigens major histocompatibility complex class II, CD1a, CD80, CD86, CD40, and CD4, so that 40% ± 9% (n = 6) of cells in culture at day 15 were CD1a+CD14−. Markers were acquired in the same sequence as on monocytes induced to differentiate with GM-CSF + IL-4. Differentiation was marked by a time-dependent increase in allostimulatory function, which, at its peak, was more potent than in cultures of DCs generated from monocytes with GM-CSF + IL-4, but was comparable on a cell-to-cell basis to that of mature monocytes cultured in flt3-ligand + c-kit-ligand + GM-CSF + IL-4 + TNF-&agr;. Both CD34+cell-derived and monocyte-derived DCs were able to process and to present tetanus toxoid and keyhole limpet hemocyanin to autologous T cells and to present major histocompatibility class I–binding peptides to CD8+cytotoxic T lymphocytes inducing interferon-&ggr; production. Altogether, these results suggest that DCs generated from CD34+cells of patients with breast cancer with flt3 ligand, c-kit ligand, GM-CSF, IL-4, and TNF-&agr; are competent antigen-presenting cells, particularly for CD8+cytotoxic T lymphocytes, and resemble mature monocyte-derived DCs in the assays described here.