New human nonimmortalized fibroblast-like cell lines were derived from various sources: from embryonic stem cells (ESCs) (the SC5-MSC and SC3a-MSC lines), from bone marrow of a 5- to 6-week-old fetus (the FetMSC line), and from foreskin of a 3-year-old child (the FRSN line). All the lines are successfully used as feeders during cultivation of human ESCs. The mean doubling time of the cell populations fluctuates depending on the line from 25.5 h in the SC5-MSC line to 38.8 h in the SC3a-MSC line. The growth curves indicate active cell proliferation of all lines. Numerical and structural karyotypical analysis has shown these lines to have a normal karyotype: 46, XX (SC5-MSC and SC3a-MSC) and 46, XY (FetMSC and FRSN). To determine the status of these lines, comparative analysis of surface markers was performed with the aid of flow cytofluorimetry, and expression of the c antigens characteristic of human mesenchymal stem cells (MSCs) was revealed: CD44, CD73, CD90, CD105, and HLA-ABC and the absence of expression of CD34 and HLA-DR. Interlinear differences in the expression level of the marker CD117 (c-kit) were revealed. Immunofluorescent and cytofluorimetric analysis of expression of surface markers and transcription factor Oct-4 that are characteristic of human ESCs has shown that, in all four lines, expression of TRA-1-60 and Oct-4 is absent, whereas in expression of SSEA-4 there are observed the interlinear differences not depending on the origin of cells. At present it is not yet clear whether the revealed interlinear differences affect essentially the functional status of mesenchymal stem cells. Immunofluorescent analysis in cells of all lines showed expression of markers of early differentiation into derivatives of three germ layers characterizing ESCs, which might possibly provide wide MSC possibilities during reparation of different tissue damages, depending on the corresponding microenvironment. The capability of cells of all lines for directed differentiation into the adipogenic and osteogenic directions was revealed.