Axotomy and neurodegenerative diseases cause corticospinal motor neuron (CSMN) degeneration. We previously showed that CNTF, NT-4 and GDNF can support CSMN survival in enriched preparations. Here we developed a fluorescence-activated cell sorting method to highly purify CSMN (97+/-4.6%). We tested the neurotrophic activities of CNTF, NT-4 and GDNF on enriched and purified CSMN preparations. Similar to their effects on enriched CSMN preparations, CNTF and GDNF sustained the survival of purified CSMN for at least 5 days with ED50 values of 1.28+/-0.46 nM and 0.59+/-0.39 nM, respectively. In contrast, NT-4 supported survival of enriched but not of purified CSMN, indicating that CNTF and GDNF sustain motor neuron survival by direct action of CSMN, while NT-4 requires accessory cells present in enriched CSMN preparations.