Human embryonic stem cells (hESCs) possess an immense potential in a variety of regenerative applications. A firm understanding of hESC mechanics, on the single cell level, may provide great insight into the role of biophysical forces in the maintenance of cellular phenotype and elucidate mechanical cues promoting differentiation along various mesenchymal lineages. Moreover, cellular biomechanics can provide an additional tool for characterizing stem cells as they follow certain differentiation lineages, and thus may aid in identifying differentiated hESCs, which are most suitable for tissue engineering. This study examined the viscoelastic properties of single undifferentiated hESCs, chondrogenically differentiated hESC subpopulations, mesenchymal stem cells (MSCs), and articular chondrocytes (ACs). hESC chondrogenesis was induced using either transforming growth factor- or knock out serum replacer as differentiation agents, and the resulting cell populations were separated based on density. All cell groups were mechanically tested using unconfined creep cytocompression. Analyses of subpopulations from all differentiation regimens resulted in a spectrum of mechanical and morphological properties spanning the range of hESCs to MSCs to ACs. Density separation was further successful in isolating cellular subpopulations with distinct mechanical properties. The instantaneous and relaxed moduli of subpopulations from differentiation regimen were statistically greater than those of undifferentiated hESCs. In addition, two subpopulations from the group were identified, which were not statistically different from native articular chondrocytes in their instantaneous and relaxed moduli, as well as their apparent viscosity. Identification of a differentiated hESC subpopulation with similar mechanical properties as native chondrocytes may provide an excellent cell source for tissue engineering applications. These cells will need to withstand any mechanical stimulation regimen employed to augment the mechanical and biochemical characteristics of the neotissue. Density separation was effective at purifying distinct populations of cells. A differentiated hESC subpopulation was identified with both similar mechanical and morphological characteristics as ACs. Future research may utilize this cell source in cartilage regeneration efforts.