Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. It has previously been reported that the NO donor sodium nitroprusside (SNP) applied to germinated tomato seeds was able to induce lateral root (LR) formation in the same way that auxin treatment does. In this paper, it is shown that NO modulates the expression of cell cycle regulatory genes in tomato pericycle cells and leads, in turn, to induced LR formation. The addition of the NO scavenger CPTIO at different time points during auxin-mediated LR development indicates that NO is required for LR primordia formation and not for LR emergence. The SNP-mediated LR promotion could be prevented by the cell cycle inhibitor olomoucine, suggesting that NO is involved in cell cycle regulation. A system was developed in which the formation of LRs was synchronized. It was based on the control of NO availability in roots by treatment with the NO scavenger CPTIO. The expression of the cell cycle regulatory genes encoding CYCA2;1, CYCA3;1, CYCD3;1, CDKA1, and the Kip-Related Protein KRP2 was studied using RT-PCR analysis in roots with synchronized and non-synchronized LR formation. NO mediates the induction of the CYCD3;1 gene and the repression of the CDK inhibitor KRP2 gene at the beginning of LR primordia formation. In addition, auxin-dependent cell cycle gene regulation was dependent on NO.