Embryonal carcinoma cells, the cancer stem cell of teratocarcinoma, provide a good model system to study various aspects of embryonic and cancer development. Abnormal karyotype not only affects the normal development and differentiation, but also boosts tumor development. As evidences suggesting the role of karyotypic abnormalities (e.g., aneuploidy and near-tetraploidy) and stem cell in tumor development are increasing, it becomes pertinent to study karyotypically abnormal embryonal carcinoma cells. We isolated a subclone, P19RG01, from long-term culture of P19 embryonal carcinoma cell line. This subclone is near-tetraploid and expresses markers of embryonal carcinoma cells. Here we show that sub-lethal heat shock induces centrosome amplification, multipolar mitosis and chromosome missegregation thereby inducing chromosomal instability by several fold. The formation of supernumerary centrosome is accompanied by a long G(2) arrest. These events lead to the generation of heterogeneous population of cells in culture. Our observations support the previous observations that selective forces (both intrinsic as well as extrinsic) play an important role in tumor progression. Moreover, we provide direct evidences which suggest chromosomal instability in cancer stem cells, using P19RG01 embryonal carcinoma cells as a model system. We propose that a minor population of chromosomally unstable cancer stem cells exists in a tumor or is generated (transient existence) in the presence of selective forces. These chromosomally unstable cancer stem cells generate a heterogeneous cancer stem cell population, which undergoes Darwinian selection, leading to selection of more malignant cancer stem cells and thereby tumor progression.