Dendritic cells (DCs) are immune cells specialized to capture, process and present antigen to T cells in order to initiate an appropriate adaptive immune response. The study of mouse DC has revealed a heterogeneous population of cells that differ in their development, surface phenotype and function. The study of human blood and spleen has shown the presence of two subsets of conventional DC including the CD1b/c 1 and CD141 1 CLEC9A 1 conventional DC (cDC) and a plasmacytoid DC (pDC) that is CD304 1 CD123 1 . Studies on these subpopulations have revealed phenotypic and functional differences that are similar to those described in the mouse. In this study, the three DC subsets have been generated in vitro from human CD34 1 precursors in the presence of fms-like tyrosine kinase 3 ligand (Flt3L) and thrombopoietin (TPO). The DC subsets so generated, including the CD1b/c 1 and CLEC9A 1 cDCs and CD123 1 pDCs, were largely similar to their blood and spleen counterparts with respect to surface phenotype, toll-like receptor and transcription factor expression, capacity to stimulate T cells, cytokine secretion and cross-presentation of antigens. This system may be utilized to study aspects of DC development and function not possible in vivo.