Since a long time, the field of stem cell biology has undergone a remarkable transformation with constant research on it and its various applications predicated to be coming into use for long term clinical cell based therapies. The present report describes extraction of mesenchymal cells from deciduous tooth and its propagation in vitro with a view to producing cells at a larger scale keeping in vitro acquisition of chromosomal aberration in mind. Pulp was extirpated from freshly exfoliated deciduous tooth and cultured within 30 minutes for colonization and harvesting of the stem cells from dental pulp. The cells had exhibited active growth. Chromosome analysis was considered for karyo typing and screening of acquired aberrations following harvesting of cultures in confluent stage and conventional cytogenetic technique. There was no evidence of abnormality in karyo type or in vitro acquisition of aberrations. The study was important to establish non-invasive collection of stem cells from biological waste (deciduous tooth), which could be monitored for chromosomal status and considered for testing of drugs/chemicals on stem cells in vitro. However, the study shall be carried out on larger sample size following passage culture for production at larger scale, which could be considered for clinical application for self or for people in need.