Like all stem cells, mesenchymal stem cells (MSC) must balance self-renewal and differentiation. Complex regulatory mechanisms are required to keep stem cells in an undifferentiated, self-renewing state and to mediate their subsequent differentiation and proliferation. In this review, we discuss how adequate numbers of MSC can be maintained in culture. In particular, we focus on identification of the cell culture conditions needed to maintain general, nonspecific potential as a stem cell over time and through replication. It would be extremely advantageous to be able to maintain MSC populations in a completely undifferentiated state and to determine and switch on specific differentiation as and when required.