Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess various advantageous properties, including self-renewal, extended proliferation potential, multi-lineage differentiation potential and capacity for differentiating into sweat gland-like cells in certain conditions. However, little is known about the effect of clinical-grade culture conditions on these properties and on the differentiative potential of hUC-MSCs. In this study, we sought to investigate the properties of hUC-MSCs expanded with animal serum free culture media (ASFCM) in order to determine their potential for differentiation into sweat gland-like cells. We found that primary cultures of hUC-MSCs could be established with ASFCM. Moreover, cells cultured in ASFCM showed vigorous proliferation comparable to those of cells grown in classical culture conditions containing fetal bovine serum (FBS). Morphology of hUC-MSCs cultured in ASFCM was comparable to those of cells grown under classical culture conditions, and hUC-MSCs grown in both of the two culture conditions tested showed the typical antigen profile of MSCs—positive for CD29, CD44, CD90, and CD105, and negative for CD34 and CD45, as expected. Chromosomal aberration assay revealed that the cells were stable after long-term culture under both culture conditions. Like normal cultured MSCs, hUC-MSCs induced under ASFCM conditions exhibited expression of the same markers (CEA, CK14 and CK19) and developmental genes (EDA and EDAR) that are characteristic of normal sweat gland cells. Taken together, our findings indicate that the classical culture medium used to differentiate hUC-MSCs into sweat gland-like cells can be replaced safely by ASFCM for clinical purposes.