Bone marrow has been considered to contain many different types of progenitor or stem cells. This study aims to establish a new strategy that provides for the rapid establishment of human clonal marrow stem cell (hcMSC) lines with a relatively small amount of bone marrow aspirate and to characterize newly generated hcMSC lines for their cell phenotype, differentiation potential, lineage-specific gene expression, and cytokine secretion. Human cMSC lines were generated with human bone marrow aspirates using a new protocol, called the subfractionation culturing method. The newly established hcMSC lines were analyzed for their cell surface epitopes by fluorescence-activated cell sorting (FACS), differentiation potential by in vitro differentiation assays, lineage-specific gene expression by RT-PCR, and cytokine secretion by enzyme-linked immunoassay (ELISA). The overall profile of the cell-surface epitopes of the newly established hcMSC lines was similar to those of the known MSCs. These hcMSC lines were capable of differentiating into multilineages with some differences in differentiation capability. In addition, these hcMSC lines secrete high levels of transforming growth factor-beta1 (TGF-beta1), leukemia inhibitory factor (LIF), TGF-alpha, and interleukein-10 (IL-10), again with some variation in each cell line. The newly designed protocol may be an efficient method to establish hcMSC lines rapidly with a relatively small amount of bone marrow sample, and these newly established hcMSC lines possess stem cell characteristics and exhibit some differences in cell-surface epitopes, differentiation potential, lineage-specific gene expression, and cytokine secretion.