Pheochromocytomas are chromaffin cell-derived neuroendocrine tumors. There is presently no cure for metastatic pheochromocytoma and no reliable way to distinguish malignant from benign tumors before the development of metastases. In order to successfully manage pheochromocytoma, it is necessary to better understand the biological determinants of tumor behavior. For this purpose, we have recently established a mouse model of metastatic pheochromocytoma using tail vein injection of mouse pheochromocytoma (MPC) cells. We optimized this model modifying the number of cells injected, length of trypsin pre-treatment, and incubation temperature and duration for the MPC cells before injection, and by serial passage and re-selection of tumors exhibiting the metastatic phenotype. We evaluated the effect of these modifications on tumor growth using serial in vivo Magnetic Resonance Imaging studies. These results show that number of cells injected, the pre-injection incubation temperature, and duration of trypsin treatment are important factors to produce faster growing, more aggressive tumors that yielded secondary metastatic lesions. Serial harvest, culture and re-selection of metastatic liver lesions produced even more aggressive pheochromocytoma cells that retained their biochemical phenotype. Microarray gene expression comparison and quantitative real-time PCR of these more aggressive cells to the MPC-parental cell line identified genes that may be important for the metastatic process.