Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45^-CD34⁺CD105⁺ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31⁺ cells, CD31^- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31⁺ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31^- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal components were up-regulated. CD31⁺ cells did not proliferate in vitro. CD45^-CD34⁺CD105⁺CD31^- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.