Multicellular organisms, from roundworms to humans, are composed of interacting individual cells that give rise to an ensemble behavior. Most current technologies enable observation of only the population-level average and often ignore the vast degree of cell heterogeneity present even in clonal populations. Single cell resolution assays of DNA, RNA, proteins, and other biomolecules can yield insights into the complex interactions present in tissues, organs, and whole organisms. Microfluidic systems facilitate single cell analyses by leveraging the micron-scale geometry for improving sensitivity, decreasing reaction time, decreasing reagents consumption, and improving parallelization and automation for high throughput. Microfluidically-generated droplets, in particular, offer extremely high scalability of reactions and straightforward single cell manipulation. This thesis presents the development of microfluidic droplet generator designs and their application for single cell analysis.Developments in microfabricated chip design presented here have resulted in versatile droplet generation tools for a wide range of applications, while novel microfabrication techniques dramatically reduced fabrication time in commercially-viable materials. A radial micropump design increased throughput per device up to 3x106 droplets per hour, allowing us to detect via digital PCR a single pathogenic E. coli O175 in a background of 105 nonpathogenic bacteria. Developing a rapid nickel mold fabrication method has facilitated prototyping and testing of microfluidic designs in thermoplastic materials in as little as 1-3 hours. These microfabrication innovations have accelerated the pace of device development to meet the needs of single cell analysis and other applications.Microfluidic technologies are opening up research paths that so far have been difficult to pursue using conventional methods. High-throughput droplet generation is used to screen purified DNA from healthy subjects exposed to carcinogens for the lymphoma-related t(14;18) chromosomal translocation with a limit of quantitation of less than 1 mutation in 107 genomes and a dynamic range of 105. We also identify unique breakpoint sites and demonstrate the ability to quantify the relative and absolute mutation frequencies within individuals for subjects with multiple mutation events. For analysis of single cell genomes, we present a novel approach for robust DNA purification and analysis using microfluidic agarose droplet encapsulation of single cells. Agarose provides a rigid yet porous shell around cells that enables purification of whole genomes for thousands of cells in parallel without the loss of single cell fidelity. We apply this method to detecting cells containing the t(14;18) translocation and sequencing two DNA targets per cell. This is extended to 9-plex forensic profiling of single cells, thus enabling analysis of complex crime scene samples with multiple contributors or samples with excessive DNA contamination.Finally, droplets are applied to investigating multiple biological parameters per cell, including growth rate, gene expression, and alternative splicing. We perform cell culture in nanoliter droplets for fast generation and monitoring of colonies originating from single cells. Colonies are subsequently assayed for telomerase hTR RNA and hTERT mRNA expression levels and hTERT splice variants. We observe a large degree of expression level bimodality for several splice variants and significant reductions in bimodality coupled with increases in alpha splicing following exposure to sub-lethal concentrations of the anti-cancer compound curcumin. Prospects for microfluidic droplets are discussed in the context of multiparameter single cell analysis as well as applications of single cell analysis to microfluidic organs-on-a-chip. Understanding basic molecular biology mechanisms from the perspective of single cells will yield insights into behavior of multicellular populations with far-reaching scientific and clinical impacts.