The macromolecular organization of the secondary wall of the cells from tree xylem is in large part responsible for the mechanical and physiological properties of wood. Modeling secondary walls of wood is difficult because information about their macromolecular architecture at the ultrastructural scale is missing. Numerous microscopic studies have provided views of the lignocellulosic composite material, but nanoscale distribution of the polymers and their interaction in muro is still not clearly understood. The intimate macromolecular organization of cell walls is defined during their differentiation. It is at the stage of wall thickening corresponding to secondary wall development that the topochemical organization and the interactions between cellulose, hemicelluloses and lignin are established.Using the conjunction of the high resolution of transmission electron microscopy (TEM) and the specificity of immunological probes directed against the main cell wall polymers, we investigated the deposition of hemicelluloses and lignins from the early stage of cambium differentiation to the mature fiber and vessel walls in growing model plants of Arabidopsis thaliana and poplar. TEM examination of differentiating cells as well as various wood and wood –derived materials and genetic plant mutants brought multiple evidence of the lamellar sub-organization of the secondary walls. Immuno-gold labeling showed that two structurally different xylan types were deposited at different stages in the wall thickening. Similarly two different types of lignin molecules were shown to be differentially polymerized at different steps of the building of the wall, lignin molecules of the condensed type being first deposited at the earliest stage of secondary thickening before the non-condensed  types. This process may be modified in response to environmental factors, as in tension wood.The spatio-temporal relationships occurring between hemicelluloses, lignin and cellulose microfibrils (CMFs) during the secondary wall development suggest that xylans with less substituted chains would be more directly interacting with CMFs than those with higher substitution patterns. It also suggests that lignin molecules of the non-condensed type have a function in bringing cohesion between the lamellae of CMFs. A model of wall assembly during secondary thickening is proposed.