Multilineage differentiation of ES cells can be demonstrated through the simple formation of embryoid bodies, which yield cells representative of all three germ layers. The cell-rich, three dimensional structure of EBs increases intercellular contact, stimulating the creation of diverse cell signalling niches that support cell differentiation to a multitude of lineages. The trajectory of differentiation within EBs can also be influenced by simple parameters such as EB size, so that manipulation of EB size can be used as an effective means to bias differentiation to desired cell types. In the absence of EB formation, ES differentiation can be directed along particular lineages by the use of defined media and/or selective passaging techniques, exemplified by numerous neural specific differentiation protocols. However, it is not always clear whether the prescribed culture conditions actively direct differentiation to the desired cell fate or affect the Ftaxilide outcome by promoting selective survival or proliferation of particular cell types. Cell fate choices of stem cells can be actively promoted by the manipulation of appropriate signalling pathways; for example exploitation of the Notch and SMAD signalling pathways can be used to direct ES cells to differentiate along the neural lineage. However, identifying the relevant signalling pathway and modulating it to direct differentiation can be difficult, due to subtle differences in cell phenotypes affecting the cellular interpretation and response to particular cues. Thus, the phenotypic output of cell differentiation is not only influenced by culture conditions and signalling pathway activity but also by the phenotype of cells in the starting population. If the starting population of cells are heterogeneous, their differentiated derivatives may also be heterogeneous. This point is especially relevant when considering the demanding problem of Pardoprunox Hydrochloride maintaining consistent in vitro culture conditions. Apparently homogeneous stem cell populations may be found to contain discrete subsets of cells that could not be initially recognised because of the absence of suitable markers.