Cell sourcing is critical in most tissue engineering applications, affecting the type and quality of extracellular matrix deposited in vitro, which makes the construct more tissue inductive once implanted in the body. Tissue specific cells represent the first choice that come to mind because they already possess the desired phenotype. However, the availability of these cells can be limited, and in the case of highly differentiated cells, their growth becomes a limiting factor. These limitations can be mitigated by the use of embryonic stem cells (ESCs). They are pluripotent, indefinitely self-renewing and thereby highly attractive for use in therapeutic applications. However, ethical controversies have posed serious problems associated with their use. Adult bone marrow mesenchymal stem cells (MSCs) avoid these ethical problems, but they are much more limited than ESCs, both in their differentiation potential and in their longevity. Thus, there is a great need for stem cells that can have great potential for therapeutic use and avoid the ethical obstacles encountered by their embryonic counterparts. Fetal tissues, such as the placenta and the umbilical cord, have been recently considered by researchers as the source of these alternative cells. Cells from the Wharton's Jelly of human umbilical cords contain high levels of transcription factors that are associated with pluripotent cells. Therefore, these Wharton's cells (WJC) can be evaluated as potential sources for different tissue engineering applications. In this study, we have assessed the osteoblastic differentiation of WJCs in 2D and 3D culture environments. Cells where harvested from the Wharton's Jelly of fresh human umbilical cords and plated on 6-well plates at passage 3. Osteogenic media, containing dexamethasone, beta-glycerophosphate and ascorbic acid, was supplemented in order to induce the differentiation. Control cultures where supplemented with alpha-MEM. Cells were also seeded on poly(L-lactic acid) (PLLA) fibrous matrices at the same inoculation density. The cultures were carried out for 1, 2 and 3 weeks. In all cultures (2D and 3D), cell growth was slower under osteogenic media than under alpha-MEM. However, in all 2D cultures, the number of cells presented a plateau after week 2. Alkaline phosphatase (ALP) activity and calcium deposition significantly increased with time, being appreciable only after week 2. When cultured in the PLLA fibrous matrices, the cells showed a continuous significant growth at all time points. ALP activity peaked after week 1, and dramatically dropped at later time points. The amount of calcium deposited displayed a continuous increase at all time points. It is important to point out that the levels of ALP activity and calcium deposition in the 3D environment were greater than those observed in 2D, even though the number of cells in the latter was greater. These findings lead us to conclude that WJCs show great potential for bone tissue engineering, and that they need a three dimensional environment to perform more efficiently.