The process of wood formation is of great interest to control and manipulate wood quality for economically important gymnosperms. A Douglas-fir tissue culture system was developed to induce differentiation of callus into tracheary elements (fibers) and to monitor xylogenesisin-vitro by a proteomics approach. Two proteomes were being compared, from an early and late stage during the fiber differentiation process. After 18 weeks in a differentiating medium 80% elongated cells and 20% of cells with advanced spiral thickening were found indicating full wood fiber differentiation. Based on 2D electrophoresis, mass spectrometric, and data analyses, it was shown that nondifferentiated callus (early stage of development) expressed proteins related to protein metabolism, cellular energy and primary cell wall metabolism. At the same time, actively differentiating wood fibers (late stage of development) expressed proteins responsible for housekeeping and stress response, but mostly proteins involved in cell wall polysaccharides biosynthesis.