Expression of differentiation markers in EB differentiation. EBs were allowed to differentiate in standard ES medium -LIF for 4 days. To determine whether each EB was consistently expressing marker genes or if these genes were only expressed in a minority of EBs, we used an RNA amplification kit to carry out one round of amplification on RNA from 16 individual EBs harvested on day 4 of differentiation, -LIF. Ten (1–10) were derived from 1000 cell hanging drops and were of similar sizes. We also harvested 6 EBs which had been allowed to aggregate randomly in solution, these were varied in size: 2 large (L1+L2), 2 medium (M1+M2) and 2 small (S1+S2). Medium EBs were the same size as those obtained from the hanging drops.