Microtubule arrays in living cells were analysed during Arabidopsis stomatal development in order to more closely define stages in the pathway and contexts where intercellular signalling might operate. Arabidopsis stomata are patterned iteratively via the orientation of an asymmetric division in a cell located next to an existing stoma. It was found that preprophase bands of microtubules (PPBs) were correctly placed away from stomata and from two types of precursor cells. This suggests that all three cell types participate in an intercellular signalling pathway that orients the division site. These and other asymmetric divisions in the pathway were preceded by a polarized cytoplasm, with the PPB around the nucleus at one end, and the vacuole at the other. PPBs before symmetric divisions of guard mother cells (GMCs) were broader than those in asymmetric divisions, and the GMC division site was marked by unusual end-wall thickenings. This work identifies an accessible system for studying cytoskeletal function and provides a foundation for analysing the role of genes involved in stomatal development.