The rapidity of the optional 90-min differentiation of Naegleria gruberi from amoebae to flagellates suggests the possibility of a free-running cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis. Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (DeltaT) and mechanical agitation, are shown to regulate differentiation synergistically with each other and with previously defined signals. A DeltaT of -10 degrees C induces complete differentiation directly in the growth environment, whereas smaller DeltaTs initiate differentiation and allow it to progress for a short time, after which the cells "hold" for up to 4 h, awaiting a stimulus to continue differentiation. Our work defines two "holdpoints," optional points in development where progress can stop, awaiting a suitable signal, while cells retain whatever intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproducibility of the differentiation, are likely to be found in other differentiating cells.