We have recently described a system for the generation of dendritic cells (DC) and Langerhans cells (LC) from defined CD34+ precursors purified from peripheral blood of healthy adult volunteers (1). This study has now been extended by the characterization of two distinct subpopulations of CD34+ cells in normal human peripheral blood as defined by the expression of the skin homing receptor cutaneous lymphocyte-associated antigen (CLA). CD34+/CLA+ cells from normal peripheral blood were found to be CD71LOW/CD11a+/CD11b+/CD49d+/ CD45RA+ whereas CD34+/CLA− cells displayed the CD71+/CD11aLOW/CD11bLOW/CD49d(+)/ CD45RALOW phenotype. To determine the differentiation pathways of these two cell populations, CD34+ cells were sorted into CLA+ and CLA− fractions, stimulated with GM-CSF and TNF-α in vitro, and then were cultured for 10 to 18 d. Similar to unfractionated CD34+ cells, the progeny of both cell populations contained sizable numbers (12–22%) of dendritically shaped, CD1a+/HLA-DR+++ cells. In addition to differences in their motility, the two dendritic cell populations generated differed from each other by the expression of LC-specific structures. Only the precursors expressing the skin homing receptor were found to differentiate into LC as evidenced by the presence of Birbeck granules. In contrast, CLA− precursor cells generated a CD1a+ DC population devoid of Birbeck granule–containing LC. Provided that comparable mechanisms as found in this study are also operative in vivo, we postulate that the topographic organization of the DC system is already determined, at least in part, at the progenitor level.