Tpx2 is a microtubule-associated protein that activates the cell-cycle kinase Aurora A and regulates the mitotic spindle. Overexpression of Tpx2 is associated with the development of different human tumors and strongly correlates with chromosomal instability. By analyzing a conditional null mutation in the mouse Tpx2 gene, we show here that Tpx2 expression is essential for spindle function and chromosome segregation in the mouse embryo. Conditional genetic ablation of Tpx2 in primary cultures resulted in deficient microtubule nucleation from DNA and aberrant spindles during prometaphase. These cells eventually exited from mitosis without chromosome segregation. In addition, Tpx2 haploinsufficiency led to the accumulation of aneuploidies in vivo and increased susceptibility to spontaneous lymphomas and lung tumors. Together, our findings indicate that Tpx2 is essential for maintaining genomic stability through its role in spindle regulation. Subtle changes in Tpx2 expression may favor tumor development in vivo.