Bone marrow (BM) mesenchymal stem cells (MSCs) represent an interesting field of research for their in vitro properties and the in vivo therapeutic applications. In the present study, we compared the clonogenic and differentiation capacity of MSCs present in three BM-derived populations-namely, the CD105(+)/CD45(-) cells, the glycophorin A (GlycoA)(-)/CD45(-) cells, and the BM mononuclear cells (BMMCs)-by growing/expanding clones from single colony-forming unit fibroblasts (CFU-F). We also quantified the Oct-4 and Nanog mRNA in the CD105(+)/CD45(-) and GlycoA(-)/CD45(-) cells to define the fraction containing more immature MSCs. We found that basic-fibroblast growth factor (bFGF) favors the long-term survival and growth of the more immature MSCs but has no significant effect on MSC clonogenic potential. CFU-F number and clone recovery were higher in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) (p < 0.0001 and p = 0.0364, respectively) cells or BMMCs (p < 0.0001 and p = 0.0007, respectively). The relative mRNA expression of Oct-4 and Nanog was significantly increased in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) cells (p < 0.0001 and p < 0.0001, respectively). No significant difference was found in the immunophenotypic characteristics and differentiation potential of clones derived from all three cellular sources. These data suggest that the CD105(+)/CD45(-) BM cell fraction is enriched in immature MSCs and, accordingly, represents an appropriate source for MSC culture initiation.